Antibody specific for CD22 and methods of use thereof

ABSTRACT

The present disclosure provides antibodies specific for an epitope present on CD22. The antibodies are useful in various treatment, diagnostic, and monitoring applications, which are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. U.S.13/942,140, filed Jul. 15, 2013, and now issued as U.S. Pat. No.9,181,343, which claims priority benefit of U.S. Provisional ApplicationNo. 61/673,630, filed Jul. 19, 2012, the contents of each of which isincorporated herein by reference in its entirety.

INTRODUCTION

CD22, a lineage-restricted B cell antigen that belongs to the Igsuperfamily, is expressed on the surface of many types of malignant Bcells, as well as on normal mature B lymphocytes.

SUMMARY OF THE INVENTION

The present disclosure provides antibodies specific for CD22. Theantibodies are useful in various treatment, diagnostic, and monitoringapplications, which are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict constructs encoding heavy (FIG. 1A) and light(FIG. 1B) chains of chimeric or humanized anti-CD22 antibodies.

FIGS. 2A-E are graphs depicting competition of humanized anti-CD22antibodies with biotinylated parental chimeric anti-CD22 antibodies forbinding to immobilized CD22.

FIGS. 3A-D depict healthy donor T cell proliferation responses to testantibodies.

FIG. 4 depicts binding of humanized anti-CD22 antibodies to Raji cells,and internalization of the antibodies by the Raji cells.

FIG. 5 depicts binding affinities of variant antibodies to human CD22.

FIGS. 6A-6D depict aggregation of humanized anti-CD22 variants.

FIGS. 7A and 7B provide an amino acid sequences of anti-CD22 heavy chain(FIG. 7A) and light chain (FIG. 7B) variable regions.

FIGS. 8A-E provide amino acid sequences of anti-CD22 antibody variantantibodies 9-20.

FIGS. 9A-C provide amino acid sequences of CD22 isoforms (Top to bottom:SEQ ID NOs:35-38).

DEFINITIONS

The terms “antibodies” and “immunoglobulin” include antibodies orimmunoglobulins of any isotype, fragments of antibodies which retainspecific binding to antigen, including, but not limited to, Fab, Fv,scFv, and Fd fragments, chimeric antibodies, humanized antibodies,single-chain antibodies, and fusion proteins comprising anantigen-binding portion of an antibody and a non-antibody protein. Theantibodies may be detectably labeled, e.g., with a radioisotope, anenzyme which generates a detectable product, a fluorescent protein, andthe like. The antibodies may be further conjugated to other moieties,such as members of specific binding pairs, e.g., biotin (member ofbiotin-avidin specific binding pair), and the like. The antibodies mayalso be bound to a solid support, including, but not limited to,polystyrene plates or beads, and the like. Also encompassed by the termare Fab′, Fv, F(ab′)₂, and or other antibody fragments that retainspecific binding to antigen, and monoclonal antibodies. An antibody maybe monovalent or bivalent.

“Antibody fragments” comprise a portion of an intact antibody, forexample, the antigen binding or variable region of the intact antibody.Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fvfragments; diabodies; linear antibodies (Zapata et al., Protein Eng.8(10): 1057-1062 (1995)); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments. Papaindigestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, a designation reflecting the abilityto crystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen combining sites and is still capable of cross-linkingantigen. “Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRS of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The “Fab” fragment also contains the constant domain of the light chainand the first constant domain (CH₁) of the heavy chain. Fab fragmentsdiffer from Fab′ fragments by the addition of a few residues at thecarboxyl terminus of the heavy chain CH₁ domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa and lambda, based on the amino acid sequences of their constantdomains. Depending on the amino acid sequence of the constant domain oftheir heavy chains, immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. In some embodiments, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains,which enables the sFv to form the desired structure for antigen binding.For a review of sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

As used herein, the term “affinity” refers to the equilibrium constantfor the reversible binding of two agents and is expressed as adissociation constant (Kd). Affinity can be at least 1-fold greater, atleast 2-fold greater, at least 3-fold greater, at least 4-fold greater,at least 5-fold greater, at least 6-fold greater, at least 7-foldgreater, at least 8-fold greater, at least 9-fold greater, at least10-fold greater, at least 20-fold greater, at least 30-fold greater, atleast 40-fold greater, at least 50-fold greater, at least 60-foldgreater, at least 70-fold greater, at least 80-fold greater, at least90-fold greater, at least 100-fold greater, or at least 1000-foldgreater, or more, than the affinity of an antibody for unrelated aminoacid sequences. Affinity of an antibody to a target protein can be, forexample, from about 100 nanomolar (nM) to about 0.1 nM, from about 100nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar(fM) or more. As used herein, the term “avidity” refers to theresistance of a complex of two or more agents to dissociation afterdilution. The terms “immunoreactive” and “preferentially binds” are usedinterchangeably herein with respect to antibodies and/or antigen-bindingfragments.

The term “binding” refers to a direct association between two molecules,due to, for example, covalent, electrostatic, hydrophobic, and ionicand/or hydrogen-bond interactions, including interactions such as saltbridges and water bridges. A subject anti-CD22 binds specifically to anepitope within a CD-22 polypeptide. Non-specific binding would refer tobinding with an affinity of less than about 10⁻⁷ M, e.g., binding withan affinity of 10⁻⁶ M, 10⁻⁵ M, 10⁻⁴ M, etc.

As used herein, the term “CDR” or “complementarity determining region”is intended to mean the non-contiguous antigen combining sites foundwithin the variable region of both heavy and light chain polypeptides.CDRs have been described by Kabat et al., J. Biol. Chem. 252:6609-6616(1977); Kabat et al., U.S. Dept. of Health and Human Services,“Sequences of proteins of immunological interest” (1991); by Chothia etal., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., J. Mol.Biol. 262:732-745 (1996), where the definitions include overlapping orsubsets of amino acid residues when compared against each other.Nevertheless, application of either definition to refer to a CDR of anantibody or grafted antibodies or variants thereof is intended to bewithin the scope of the term as defined and used herein. The amino acidresidues which encompass the CDRs as defined by each of the above citedreferences are set forth below in Table 1 as a comparison.

TABLE 1 CDR Definitions Kabat¹ Chothia² MacCallum³ V_(H) CDR1 31-3526-32 30-35 V_(H) CDR2 50-65 53-55 47-58 V_(H) CDR3  95-102  96-101 93-101 V_(L) CDR1 24-34 26-32 30-36 V_(L) CDR2 50-56 50-52 46-55 V_(L)CDR3 89-97 91-96 89-96 ¹Residue numbering follows the nomenclature ofKabat et al., supra ²Residue numbering follows the nomenclature ofChothia et al., supra ³Residue numbering follows the nomenclature ofMacCallum et al., supra

As used herein, the term “framework” when used in reference to anantibody variable region is intended to mean all amino acid residuesoutside the CDR regions within the variable region of an antibody. Avariable region framework is generally a discontinuous amino acidsequence between about 100-120 amino acids in length but is intended toreference only those amino acids outside of the CDRs. As used herein,the term “framework region” is intended to mean each domain of theframework that is separated by the CDRs.

An “isolated” antibody is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In some embodiments, the antibody will bepurified (1) to greater than 90%, greater than 95%, or greater than 98%,by weight of antibody as determined by the Lowry method, for example,more than 99% by weight, (2) to a degree sufficient to obtain at least15 residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (3) to homogeneity by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing ornonreducing conditions using Coomassie blue or silver stain. Isolatedantibody includes the antibody in situ within recombinant cells since atleast one component of the antibody's natural environment will not bepresent. In some instances, isolated antibody will be prepared by atleast one purification step.

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse affectattributable to the disease. “Treatment,” as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease but has not yet been diagnosed ashaving it; (b) inhibiting the disease, i.e., arresting its development;and (c) relieving the disease, i.e., causing regression of the disease.

The terms “individual,” “subject,” “host,” and “patient,” usedinterchangeably herein, refer to a mammal, including, but not limitedto, murines (rats, mice), non-human primates, humans, canines, felines,ungulates (e.g., equines, bovines, ovines, porcines, caprines), etc.

A “therapeutically effective amount” or “efficacious amount” refers tothe amount of a subject anti-CD22 Ab that, when administered to a mammalor other subject for treating a disease, is sufficient to effect suchtreatment for the disease. The “therapeutically effective amount” willvary depending on the anti-CD22 Ab, the disease and its severity and theage, weight, etc., of the subject to be treated.

A “biological sample” encompasses a variety of sample types obtainedfrom an individual and can be used in a diagnostic or monitoring assay.The definition encompasses blood and other liquid samples of biologicalorigin, solid tissue samples such as a biopsy specimen or tissuecultures or cells derived therefrom and the progeny thereof. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such aspolynucleotides. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, serum, plasma, biological fluid, and tissue samples. In somecases, a biological sample will include B cells.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anantibody” includes a plurality of such antibodies and reference to “theCDR” includes reference to one or more CDRs and equivalents thereofknown to those skilled in the art, and so forth. It is further notedthat the claims may be drafted to exclude any optional element. As such,this statement is intended to serve as antecedent basis for use of suchexclusive terminology as “solely,” “only” and the like in connectionwith the recitation of claim elements, or use of a “negative”limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

The present disclosure provides antibodies specific for CD22. Theantibodies are useful in various treatment, diagnostic, and monitoringapplications, which are also provided.

Antibodies

A subject antibody specifically binds a CD22 polypeptide, where theepitope comprises amino acid residues within a CD22 antigen (e.g.,within amino acids 1 to 847, within amino acids 1-759, within aminoacids 1-751, or within amino acids 1-670, of a CD22 amino acid sequencedepicted in FIGS. 9A-C).

The CD22 epitope can be formed by a polypeptide having at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from about 500 aminoacids to about 670 amino acids of the human CD22 isoform 4 amino acidsequence depicted in FIGS. 9A-C. The CD22 epitope can be formed by apolypeptide having at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to acontiguous stretch of from about 500 amino acids to about 751 aminoacids of the human CD22 isoform 3 amino acid sequence depicted in FIGS.9A-C. The CD22 epitope can be formed by a polypeptide having at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity to a contiguous stretch of from about 500amino acids to about 759 amino acids of the human CD22 isoform 2 aminoacid sequence depicted in FIGS. 9A-C. The CD22 epitope can be formed bya polypeptide having at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or 100%, amino acid sequence identity to acontiguous stretch of from about 500 amino acids to about 847 aminoacids of the human CD22 isoform 1 amino acid sequence depicted in FIGS.9A-C.

A subject antibody exhibits high affinity binding to CD22. For example,a subject antibody binds to CD22 with an affinity of at least about 10⁻⁷M, at least about 10⁻⁸ M, at least about 10⁻⁹ M, at least about 10⁻¹⁰ M,at least about 10⁻¹¹ M, or at least about 10⁻¹² M, or greater than 10⁻¹²M. A subject antibody binds to an epitope present on CD22 with anaffinity of from about 10⁻⁷ M to about 10⁻⁸ M, from about 10⁻⁸ M toabout 10⁻⁹ M, from about 10⁻⁹ M to about 10⁻¹⁰ M, from about 10⁻¹⁰ M toabout 10⁻¹¹ M, or from about 10⁻¹¹ M to about 10⁻¹² M, or greater than10⁻¹² M. An anti-CD22 antibody of the present disclosure can in somecases induce apoptosis in a cell that expresses CD22 on its cellsurface.

A “CD22 antigen” or “CD22 polypeptide” can comprises an amino acidsequence having at least about 75%, at least about 80%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to a contiguous stretch of from about500 amino acids (aa) to about 847 aa (isoform 1), to about 759 aa(isoform 2), to about 751 aa (isoform 3), or to about 670 aa (isoform 4)of a CD22 isoform 1, 2, 3, or 4 amino acid sequence depicted in FIGS.9A-C.

The term “antibody” refers to a protein comprising one or more (e.g.,one or two) heavy chain variable regions (VH) and/or one or more (e.g.,one or two) light chain variable regions (VL), or subfragments thereofcapable of binding an epitope. The VH and VL regions can be furthersubdivided into regions of hypervariability, termed “complementaritydetermining regions (CDR)”, interspersed with regions that are moreconserved, termed “framework regions (FR)”. The extent of the FR andCDRs has been precisely defined (see, Kabat, et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242; Chothia et al.(1987) J. Mol. Biol. 196: 901-917). A VH can comprise three CDRs andfour FRs arranged from N-terminus to C-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Similarly, a VL can comprise threeCDRs and four FRs arranged from N-terminus to C-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The VH or VL chain of an antibody can further include all or part of aheavy or light chain constant region, to thereby form a heavy or lightimmunoglobulin chain, respectively. In one embodiment, the antibody is atetramer of two heavy and two light chains, wherein the heavy and lightchains are interconnected by, for example, disulphide bonds. The heavychain constant region is comprised of three domains, CH1, CH2 and CH3.The light chain constant region is comprised of one domain, CL. Thevariable regions of the heavy and light chains comprise binding regionsthat interact with antigen. The constant regions of the antibodiestypically mediate the binding of the antibody to host tissues andfactors, including various cells of the immune system and the firstcomponent of the complement system. The term “antibody” includes intactimmunoglobulins of types IgA, IgG, IgE, IgD, IgM and subtypes thereof.In some embodiments, a subject antibody is an IgG isotype. In someembodiments, a subject antibody is an IgG1 isotype.

As used herein the term “immunoglobulin” refers to a protein consistingof one or more polypeptides substantially encoded by immunoglobulingenes. The recognized human immunoglobulin genes include the kappa,lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta,epsilon and mu constant region genes; and numerous immunoglobulinvariable region genes. Full-length immunoglobulin light chains (about 25kD or 214 amino acids) are encoded by a variable region gene at theN-terminus (about 110 amino acids) and a kappa or lambda constant regionat the C-terminus. Full-length immunoglobulin heavy chains (about 50 kDor 446 amino acids) are encoded by a variable region gene at theN-terminus (about 116 amino acids) and one of the other aforementionedconstant region genes at the C-terminus, e.g. gamma (encoding about 330amino acids). In some embodiments, a subject antibody comprisesfull-length immunoglobulin heavy chain and a full-length immunoglobulinlight chain.

In some embodiments, a subject antibody does not comprise a full-lengthimmunoglobulin heavy chain and a full-length immunoglobulin light chain,and instead comprises antigen-binding fragments of a full-lengthimmunoglobulin heavy chain and a full-length immunoglobulin light chain.In some embodiments, the antigen-binding fragments are contained onseparate polypeptide chains; in other embodiments, the antigen-bindingfragments are contained within a single polypeptide chain. The term“antigen-binding fragment” refers to one or more fragments of afull-length antibody that are capable of specifically binding to CD22,as described above. Examples of binding fragments include (i) a Fabfragment (a monovalent fragment consisting of the VL, VH, CL and CH1domains; (ii) a F(ab′)₂ fragment (a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment (consisting of the VH and CH1 domains); (iv) a Fv fragment(consisting of the VH and VL domains of a single arm of an antibody);(v) a dAb fragment (consisting of the VH domain); (vi) an isolated CDR;(vii) a single chain Fv (scFv) (consisting of the VH and VL domains of asingle arm of an antibody joined by a synthetic linker using recombinantmeans such that the VH and VL domains pair to form a monovalentmolecule); (viii) diabodies (consisting of two scFvs in which the VH andVL domains are joined such that they do not pair to form a monovalentmolecule; the VH of each one of the scFv pairs with the VL domain of theother scFv to form a bivalent molecule); (ix) bi-specific antibodies(consisting of at least two antigen binding regions, each region bindinga different epitope). In some embodiments, a subject antibody fragmentis a Fab fragment. In some embodiments, a subject antibody fragment is asingle-chain antibody (scFv). In some embodiments, a subject antibody isa recombinant or modified antibody, e.g., a chimeric, humanized,deimmunized or an in vitro generated antibody. The term “recombinant” or“modified” antibody as used herein is intended to include all antibodiesthat are prepared, expressed, created, or isolated by recombinant means,such as (i) antibodies expressed using a recombinant expression vectortransfected into a host cell; (ii) antibodies isolated from arecombinant, combinatorial antibody library; (iii) antibodies isolatedfrom an animal (e.g. a mouse) that is transgenic for humanimmunoglobulin genes; or (iv) antibodies prepared, expressed, created,or isolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinantantibodies include humanized, CDR grafted, chimeric, deimmunized, and invitro generated antibodies; and can optionally include constant regionsderived from human germline immunoglobulin sequences.

In some embodiments, a subject antibody comprises: a) a heavy chaincomprising a VH region having the amino acid sequenceEVQLVESGGGLVKPGGSLX¹LSCAASGFAFSIYDMSWVRQAPGKGLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNX²LYLQMX³SLRAEDTAMYYCARHSGYGSSYGVLFAYWG QGTLVTVSS(SEQ ID NO:1), where X¹ is K (Lys) or R (Arg); X² is S (Ser) or T (Thr);and X³ is N (Asn) or S (Ser); and b) an immunoglobulin light chain.

A light chain can have any suitable V_(L) amino acid sequence, so longas the resulting antibody binds specifically to CD22.

Exemplary V_(L) amino acid sequences include:

(SEQ ID NO: 7; VK1) DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTSILHSGVPSRFSGSGSGTDYTLTISSLQQEDFATYFCQQGNTL PWTFGGGTKVEIK;(SEQ ID NO: 8; VK2) DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTSILHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTL PWTFGGGTKVEIK; and(SEQ ID NO: 9; VK4) DIQMTQSPSSVSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTL PWTFGGGTKVEIK.

Thus, e.g., a subject anti-CD22 antibody can comprise: a) a heavy chaincomprising a VH region having the amino acid sequence set forth in SEQID NO:1); and a light chain comprising the VL region of VK1. In othercases, a subject anti-CD22 antibody can comprise: a) a heavy chaincomprising a VH region having the amino acid sequence set forth in SEQID NO:1); and a light chain comprising the VL region of VK2. In stillother cases, a subject anti-CD22 antibody can comprise: a) a heavy chaincomprising a VH region having the amino acid sequence set forth in SEQID NO:1); and a light chain comprising the VL region of VK4.

In some instances, a subject anti-CD22 antibody comprises: a) animmunoglobulin light chain comprising the amino acid sequenceDIQMTQSPSSX¹SASVGDRVTITCRASQDISNYLNWYQQKPGKAX²KLLIYYTSILHSGVPSRFSGSGSGTDYTLTISSLQX³EDFATYFCQQGNTLPWTFGGGTKVEIK (SEQ ID NO:2), whereX¹ is L (Leu) or V (Val); X² is V (Val) or P (Pro); and X³ is Q (Gln) orP (Pro); and b) an immunoglobulin heavy chain. The heavy chain cancomprise an amino acid sequence selected from:

(SEQ ID NO: 3; VH3) EVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQAPGKGLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNTLYLQMSSLRAEDTAMYYCARHSGYGSSYGVLFAYWGQGTLVTVSS; (SEQ ID NO: 4; VH4)EVQLVESGGGLVKPGGSLRLSCAASGFAFSIYDMSWVRQAPGKGLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNSLYLQMSSLRAEDTAMYYCARHSGYGSSYGVLFAYWGQGTLVTVSS; (SEQ ID NO: 5; VH5)EVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQAPGKGLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAMYYCARHSGYGSSYGVLFAYWGQGTLVTVSS; and (SEQ ID NO: 6; VH6)EVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQAPGKGLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNSLYLQMSSLRAEDTAMYYCARHSGYGSSYGVLFAYWGQGTLVTVSS.

Linkers suitable for use a subject antibody include “flexible linkers.”If present, the linker molecules are generally of sufficient length topermit some flexible movement between linked regions. The linkermolecules are generally about 6-50 atoms long. The linker molecules mayalso be, for example, aryl acetylene, ethylene glycol oligomerscontaining 2-10 monomer units, diamines, diacids, amino acids, orcombinations thereof. Other linker molecules which can bind topolypeptides may be used in light of this disclosure.

A subject antibody can be “humanized.” The term “humanized antibody”refers to an antibody comprising at least one chain comprising variableregion framework residues substantially from a human antibody chain(referred to as the acceptor immunoglobulin or antibody) and at leastone CDR substantially from a mouse antibody, (referred to as the donorimmunoglobulin or antibody). See, Queen et al., Proc. Natl. Acad. Sci.USA 86:10029 10033 (1989), U.S. Pat. No. 5,530,101, U.S. Pat. No.5,585,089, U.S. Pat. No. 5,693,761, WO 90/07861, and U.S. Pat. No.5,225,539. The constant region(s), if present, can also be substantiallyor entirely from a human immunoglobulin. Methods of making humanizedantibodies are known in the art. See, e.g., U.S. Pat. No. 7,256,273.

The substitution of mouse CDRs into a human variable domain frameworkcan result in retention of their correct spatial orientation where,e.g., the human variable domain framework adopts the same or similarconformation to the mouse variable framework from which the CDRsoriginated. This can be achieved by obtaining the human variable domainsfrom human antibodies whose framework sequences exhibit a high degree ofsequence identity with the murine variable framework domains from whichthe CDRs were derived. The heavy and light chain variable frameworkregions can be derived from the same or different human antibodysequences. The human antibody sequences can be the sequences ofnaturally occurring human antibodies or can be consensus sequences ofseveral human antibodies. See Kettleborough et al., Protein Engineering4:773 (1991); Kolbinger et al., Protein Engineering 6:971 (1993).

Having identified the complementarity determining regions of the murinedonor immunoglobulin and appropriate human acceptor immunoglobulins, thenext step is to determine which, if any, residues from these componentsshould be substituted to optimize the properties of the resultinghumanized antibody. In general, substitution of human amino acidresidues with murine should be minimized, because introduction of murineresidues increases the risk of the antibody eliciting ahuman-anti-mouse-antibody (HAMA) response in humans. Art-recognizedmethods of determining immune response can be performed to monitor aHAMA response in a particular patient or during clinical trials.Patients administered humanized antibodies can be given animmunogenicity assessment at the beginning and throughout theadministration of said therapy. The HAMA response is measured, forexample, by detecting antibodies to the humanized therapeutic reagent,in serum samples from the patient using a method known to one in theart, including surface plasmon resonance technology (BIACORE) and/orsolid-phase ELISA analysis. In many embodiments, a subject humanizedantibody does not substantially elicit a HAMA response in a humansubject.

Certain amino acids from the human variable region framework residuesare selected for substitution based on their possible influence on CDRconformation and/or binding to antigen. The unnatural juxtaposition ofmurine CDR regions with human variable framework region can result inunnatural conformational restraints, which, unless corrected bysubstitution of certain amino acid residues, lead to loss of bindingaffinity.

The selection of amino acid residues for substitution can be determined,in part, by computer modeling. Computer hardware and software forproducing three-dimensional images of immunoglobulin molecules are knownin the art. In general, molecular models are produced starting fromsolved structures for immunoglobulin chains or domains thereof. Thechains to be modeled are compared for amino acid sequence similaritywith chains or domains of solved three-dimensional structures, and thechains or domains showing the greatest sequence similarity is/areselected as starting points for construction of the molecular model.Chains or domains sharing at least 50% sequence identity are selectedfor modeling, and preferably those sharing at least 60%, 70%, 80%, 90%sequence identity or more are selected for modeling. The solved startingstructures are modified to allow for differences between the actualamino acids in the immunoglobulin chains or domains being modeled, andthose in the starting structure. The modified structures are thenassembled into a composite immunoglobulin. Finally, the model is refinedby energy minimization and by verifying that all atoms are withinappropriate distances from one another and that bond lengths and anglesare within chemically acceptable limits. CDR and framework regions areas defined by Kabat, Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md., 1987 and 1991). Analternative structural definition has been proposed by Chothia et al.,J. Mol. Biol. 196:901 (1987); Nature 342:878 (1989); and J. Mol. Biol.186:651 (1989) (collectively referred to as “Chothia”). When frameworkresidues, as defined by Kabat, supra, constitute structural loopresidues as defined by Chothia, supra, the amino acids present in themouse antibody may be selected for substitution into the humanizedantibody. Residues which are “adjacent to a CDR region” include aminoacid residues in positions immediately adjacent to one or more of theCDRs in the primary sequence of the humanized immunoglobulin chain, forexample, in positions immediately adjacent to a CDR as defined by Kabat,or a CDR as defined by Chothia (See e.g., Chothia and Lesk J M B 196:901(1987)). These amino acids are particularly likely to interact with theamino acids in the CDRs and, if chosen from the acceptor, to distort thedonor CDRs and reduce affinity. Moreover, the adjacent amino acids mayinteract directly with the antigen (Amit et al., Science, 233:747(1986)) and selecting these amino acids from the donor may be desirableto keep all the antigen contacts that provide affinity in the originalantibody.

In some embodiments, a subject antibody comprises scFv multimers. Forexample, in some embodiments, a subject antibody is an scFv dimer (e.g.,comprises two tandem scFv (scFv₂)), an scFv trimer (e.g., comprisesthree tandem scFv (scFv₃)), an scFv tetramer (e.g., comprises fourtandem scFv (scFv₄)), or is a multimer of more than four scFv (e.g., intandem). The scFv monomers can be linked in tandem via linkers of fromabout 2 amino acids to about 10 amino acids in length, e.g., 2 aa, 3 aa,4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa in length. Suitable linkersinclude, e.g., (Gly)_(x), where x is an integer from 2 to 10. Othersuitable linkers are those discussed above. In some embodiments, each ofthe scFv monomers in a subject scFV multimer is humanized, as describedabove.

In some embodiments, a subject antibody comprises a constant region ofan immunoglobulin (e.g., an Fc region). The Fc region, if present, canbe a human Fc region. If constant regions are present, the antibody cancontain both light chain and heavy chain constant regions. Suitableheavy chain constant region include CHL hinge, CH2, CH3, and CH4regions. The antibodies described herein include antibodies having alltypes of constant regions, including IgM, IgG, IgD, IgA and IgE, and anyisotype, including IgG1, IgG2, IgG3 and IgG4. An example of a suitableheavy chain Fc region is a human isotype IgG1 Fc. Light chain constantregions can be lambda or kappa. A subject antibody (e.g., a subjecthumanized antibody) can comprise sequences from more than one class orisotype. Antibodies can be expressed as tetramers containing two lightand two heavy chains, as separate heavy chains, light chains, as Fab,Fab′ F(ab′)2, and Fv, or as single chain antibodies in which heavy andlight chain variable domains are linked through a spacer.

In some embodiments, a subject antibody comprises a free thiol (—SH)group at the carboxyl terminus, where the free thiol group can be usedto attach the antibody to a second polypeptide (e.g., another antibody,including a subject antibody), a scaffold, a carrier, etc.

In some embodiments, a subject antibody comprises one or morenon-naturally occurring amino acids. In some embodiments, thenon-naturally encoded amino acid comprises a carbonyl group, an acetylgroup, an aminooxy group, a hydrazine group, a hydrazide group, asemicarbazide group, an azide group, or an alkyne group. See, e.g., U.S.Pat. No. 7,632,924 for suitable non-naturally occurring amino acids.Inclusion of a non-naturally occurring amino acid can provide forlinkage to a polymer, a second polypeptide, a scaffold, etc. Forexample, a subject antibody linked to a water-soluble polymer can bemade by reacting a water-soluble polymer (e.g., PEG) that comprises acarbonyl group to a subject antibody that comprises a non-naturallyencoded amino acid that comprises an aminooxy, hydrazine, hydrazide orsemicarbazide group. As another example, a subject antibody linked to awater-soluble polymer can be made by reacting a subject antibody thatcomprises an alkyne-containing amino acid with a water-soluble polymer(e.g., PEG) that comprises an azide moiety; in some embodiments, theazide or alkyne group is linked to the PEG molecule through an amidelinkage. A “non-naturally encoded amino acid” refers to an amino acidthat is not one of the 20 common amino acids or pyrrolysine orselenocysteine. Other terms that may be used synonymously with the term“non-naturally encoded amino acid” are “non-natural amino acid,”“unnatural amino acid,” “non-naturally-occurring amino acid,” andvariously hyphenated and non-hyphenated versions thereof. The term“non-naturally encoded amino acid” also includes, but is not limited to,amino acids that occur by modification (e.g. post-translationalmodifications) of a naturally encoded amino acid (including but notlimited to, the 20 common amino acids or pyrrolysine and selenocysteine)but are not themselves naturally incorporated into a growing polypeptidechain by the translation complex. Examples of suchnon-naturally-occurring amino acids include, but are not limited to,N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, andO-phosphotyrosine. The present disclosure also provides anti-CD22antibodies having an attached moiety of interest, e.g. a detectablelabel, drug, half-life-extending moiety, and the like. Modification ofantibodies can be accomplished by a variety of synthetic and/orrecombinant methods. The moiety or moieties attached to an antibody canprovide for one or more of a wide variety of functions or features.Exemplary moieties include detectable labels (e.g., dye labels (e.g.,chromophores, fluorophores), biophysical probes (spin labels, nuclearmagnetic resonance (NMR) probes), Förster Resonance Energy Transfer(FRET)-type labels (e.g., at least one member of a FRET pair, includingat least one member of a fluorophore/quencher pair), BioluminescenceResonance Energy Transfer (BRET)-type labels (e.g., at least one memberof a BRET pair), immunodetectable tags (e.g., FLAG, His(6), and thelike); water soluble polymers (e.g., PEGylation); purification tags(e.g., to facilitate isolation by affinity chromatography (e.g.,attachment of a FLAG epitope; membrane localization domains (e.g.,lipids or glycophosphatidylinositol (GPI)-type anchors); immobilizationtags (e.g., to facilitate attachment of the polypeptide to a surface,including selective attachment); drugs (e.g., to facilitate drugtargeting, e.g., through attachment of the drug to an antibody); and thelike.

In some embodiments, a subject antibody is linked (e.g., covalentlylinked) to a polymer (e.g., a polymer other than a polypeptide).Suitable polymers include, e.g., biocompatible polymers, andwater-soluble biocompatible polymers. Suitable polymers includesynthetic polymers and naturally-occurring polymers. Suitable polymersinclude, e.g., substituted or unsubstituted straight or branched chainpolyalkylene, polyalkenylene or polyoxyalkylene polymers or branched orunbranched polysaccharides, e.g. a homo- or hetero-polysaccharide.Suitable polymers include, e.g., ethylene vinyl alcohol copolymer(commonly known by the generic name EVOH or by the trade name EVAL);polybutylmethacrylate; poly(hydroxyvalerate); poly(L-lactic acid);polycaprolactone; poly(lactide-co-glycolide); poly(hydroxybutyrate);poly(hydroxybutyrate-co-valerate); polydioxanone; polyorthoester;polyanhydride; poly(glycolic acid); poly(D,L-lactic acid); poly(glycolicacid-co-trimethylene carbonate); polyphosphoester; polyphosphoesterurethane; poly(amino acids); cyanoacrylates; poly(trimethylenecarbonate); poly(iminocarbonate); copoly(ether-esters) (e.g.,poly(ethylene oxide)-poly(lactic acid) (PEO/PLA) co-polymers);polyalkylene oxalates; polyphosphazenes; biomolecules, such as fibrin,fibrinogen, cellulose, starch, collagen and hyaluronic acid;polyurethanes; silicones; polyesters; polyolefins; polyisobutylene andethylene-alphaolefin copolymers; acrylic polymers and copolymers; vinylhalide polymers and copolymers, such as polyvinyl chloride; polyvinylethers, such as polyvinyl methyl ether; polyvinylidene halides, such aspolyvinylidene fluoride and polyvinylidene chloride; polyacrylonitrile;polyvinyl ketones; polyvinyl aromatics, such as polystyrene; polyvinylesters, such as polyvinyl acetate; copolymers of vinyl monomers witheach other and olefins, such as ethylene-methyl methacrylate copolymers,acrylonitrile-styrene copolymers, ABS resins, and ethylene-vinyl acetatecopolymers; polyamides, such as Nylon 66 and polycaprolactam; alkydresins; polycarbonates; polyoxymethylenes; polyimides; polyethers; epoxyresins; polyurethanes; rayon; rayon-triacetate; cellulose; celluloseacetate; cellulose butyrate; cellulose acetate butyrate; cellophane;cellulose nitrate; cellulose propionate; cellulose ethers; amorphousTeflon; poly(ethylene glycol); and carboxymethyl cellulose.

Suitable synthetic polymers include unsubstituted and substitutedstraight or branched chain poly(ethyleneglycol), poly(propyleneglycol)poly(vinylalcohol), and derivatives thereof, e.g., substitutedpoly(ethyleneglycol) such as methoxypoly(ethyleneglycol), andderivatives thereof. Suitable naturally-occurring polymers include,e.g., albumin, amylose, dextran, glycogen, and derivatives thereof.

Suitable polymers can have an average molecular weight in a range offrom 500 Da to 50000 Da, e.g., from 5000 Da to 40000 Da, or from 25000to 40000 Da. For example, in some embodiments, where a subject antibodycomprises a poly(ethylene glycol) (PEG) or methoxypoly(ethyleneglycol)polymer, the PEG or methoxypoly(ethyleneglycol) polymer can have amolecular weight in a range of from about 0.5 kiloDaltons (kDa) to 1kDa, from about 1 kDa to 5 kDa, from 5 kDa to 10 kDa, from 10 kDa to 25kDa, from 25 kDa to 40 kDa, or from 40 kDa to 60 kDa.

As noted above, in some embodiments, a subject antibody is covalentlylinked to a PEG polymer. In some embodiments, a subject scFv multimer iscovalently linked to a PEG polymer. Methods and reagents suitable forPEGylation of a protein are well known in the art and may be found in,e.g., U.S. Pat. No. 5,849,860. PEG suitable for conjugation to a proteinis generally soluble in water at room temperature, and has the generalformula R(O—CH₂—CH₂)_(n)O—R, where R is hydrogen or a protective groupsuch as an alkyl or an alkanol group, and where n is an integer from 1to 1000. Where R is a protective group, it generally has from 1 to 8carbons.

The PEG conjugated to the subject antibody can be linear. The PEGconjugated to the subject protein may also be branched. Branched PEGderivatives such as those described in U.S. Pat. No. 5,643,575,“star-PEG's” and multi-armed PEG's such as those described in ShearwaterPolymers, Inc. catalog “Polyethylene Glycol Derivatives 1997-1998.” StarPEGs are described in the art including, e.g., in U.S. Pat. No.6,046,305.

A subject antibody can be glycosylated, e.g., a subject antibody cancomprise a covalently linked carbohydrate or polysaccharide moiety.Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used. Glycosylation can be accomplished by,for example, recombination production in a host cell having the desiredglycosylation machinery.

Addition of glycosylation sites to an antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).Similarly, removal of glycosylation sites can be accomplished by aminoacid alteration within the native glycosylation sites of an antibody.

A subject antibody can be covalently linked to a second moiety (e.g., alipid, a polypeptide other than a subject antibody, a synthetic polymer,a carbohydrate, and the like) using for example, glutaraldehyde, ahomobifunctional cross-linker, or a heterobifunctional cross-linker.Glutaraldehyde cross-links polypeptides via their amino moieties.Homobifunctional cross-linkers (e.g., a homobifunctional imidoester, ahomobifunctional N-hydroxysuccinimidyl (NHS) ester, or ahomobifunctional sulfhydryl reactive cross-linker) contain two or moreidentical reactive moieties and can be used in a one step reactionprocedure in which the cross-linker is added to a solution containing amixture of the polypeptides to be linked. Homobifunctional NHS ester andimido esters cross-link amine containing polypeptides. In a mildalkaline pH, imido esters react only with primary amines to formimidoamides, and overall charge of the cross-linked polypeptides is notaffected. Homobifunctional sulfhydryl reactive cross-linkers includesbismaleimidhexane (BMH), 1,5-difluoro-2,4-dinitrobenzene (DFDNB), and1,4-di-(3′,2′-pyridyldithio) propinoamido butane (DPDPB).

Heterobifunctional cross-linkers have two or more different reactivemoieties (e.g., amine reactive moiety and a sulfhydryl-reactive moiety)and are cross-linked with one of the polypeptides via the amine orsulfhydryl reactive moiety, then reacted with the other polypeptide viathe non-reacted moiety. Multiple heterobifunctional haloacetylcross-linkers are available, as are pyridyl disulfide cross-linkers.Carbodiimides are a classic example of heterobifunctional cross-linkingreagents for coupling carboxyls to amines, which results in an amidebond.

A subject antibody can be immobilized on a solid support. Suitablesupports are well known in the art and comprise, inter alia,commercially available column materials, polystyrene beads, latex beads,magnetic beads, colloid metal particles, glass and/or silicon chips andsurfaces, nitrocellulose strips, nylon membranes, sheets, duracytes,wells of reaction trays (e.g., multi-well plates), plastic tubes, etc. Asolid support can comprise any of a variety of substances, including,e.g., glass, polystyrene, polyvinyl chloride, polypropylene,polyethylene, polycarbonate, dextran, nylon, amylose, natural andmodified celluloses, polyacrylamides, agaroses, and magnetite. Suitablemethods for immobilizing a subject antibody onto a solid support arewell known and include, but are not limited to ionic, hydrophobic,covalent interactions and the like. Solid supports can be soluble orinsoluble, e.g., in aqueous solution. In some embodiments, a suitablesolid support is generally insoluble in an aqueous solution.

A subject antibody can in some embodiments comprise a detectable label.Suitable detectable labels include any composition detectable byspectroscopic, photochemical, biochemical, immunochemical, electrical,optical or chemical means. Suitable include, but are not limited to,magnetic beads (e.g. Dynabeads™), fluorescent dyes (e.g., fluoresceinisothiocyanate, texas red, rhodamine, a green fluorescent protein, a redfluorescent protein, a yellow fluorescent protein, and the like),radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, and others commonlyused in an enzyme-linked immunosorbent assay (ELISA)), and colorimetriclabels such as colloidal gold or colored glass or plastic (e.g.polystyrene, polypropylene, latex, etc.) beads.

In some embodiments, a subject antibody comprises a contrast agent or aradioisotope, where the contrast agent or radioisotope is one that issuitable for use as a detectable label, e.g., in imaging, e.g., imagingprocedures carried out on humans. Non-limiting examples of labelsinclude radioisotope such as ¹²³¹I (iodine), ¹⁸F (fluorine), ⁹⁹Tc(technetium), ¹¹¹In (indium), and ⁶⁷Ga (gallium), and contrast agentsuch as gadolinium (Gd), dysprosium, and iron. Radioactive Gd isotopes(¹⁵³Gd) also are available and suitable for imaging procedures innon-human mammals. A subject antibody can be labeled using standardtechniques. For example, a subject antibody can be iodinated usingchloramine T or 1,3,4,6-tetrachloro-3α,6α-dephenylglycouril. Forfluorination, fluorine is added to a subject antibody during thesynthesis by a fluoride ion displacement reaction. See, Muller-Gartner,H., TIB Tech., 16:122-130 (1998) and Saji, H., Crit. Rev. Ther. DrugCarrier Syst., 16(2):209-244 (1999) for a review of synthesis ofproteins with such radioisotopes. A subject antibody can also be labeledwith a contrast agent through standard techniques. For example, asubject antibody can be labeled with Gd by conjugating low molecular Gdchelates such as Gd diethylene triamine pentaacetic acid (GdDTPA) or Gdtetraazacyclododecanetetraacetic (GdDOTA) to the antibody. See, Caravanet al., Chem. Rev. 99:2293-2352 (1999) and Lauffer et al., J. Magn.Reson. Imaging, 3:11-16 (1985). A subject antibody can be labeled withGd by, for example, conjugating polylysine-Gd chelates to the antibody.See, for example, Curtet et al., Invest. Radiol., 33(10):752-761 (1998).Alternatively, a subject antibody can be labeled with Gd by incubatingparamagnetic polymerized liposomes that include Gd chelator lipid withavidin and biotinylated antibody. See, for example, Sipkins et al.,Nature Med., 4:623-626 (1998).

Suitable fluorescent proteins that can be linked to a subject antibodyinclude, but are not limited to, a green fluorescent protein fromAequoria victoria or a mutant or derivative thereof e.g., as describedin U.S. Pat. Nos. 6,066,476; 6,020,192; 5,985,577; 5,976,796; 5,968,750;5,968,738; 5,958,713; 5,919,445; 5,874,304; e.g., Enhanced GFP, manysuch GFP which are available commercially, e.g., from Clontech, Inc.; ared fluorescent protein; a yellow fluorescent protein; any of a varietyof fluorescent and colored proteins from Anthozoan species, as describedin, e.g., Matz et al. (1999) Nature Biotechnol. 17:969-973; and thelike.

A subject antibody will in some embodiments comprise a “radiopaque”label, e.g. a label that can be easily visualized using for examplex-rays. Radiopaque materials are well known to those of skill in theart. The most common radiopaque materials include iodide, bromide orbarium salts. Other radiopaque materials are also known and include, butare not limited to organic bismuth derivatives (see, e.g., U.S. Pat. No.5,939,045), radiopaque multiurethanes (see U.S. Pat. No. 5,346,981),organobismuth composites (see, e.g., U.S. Pat. No. 5,256,334),radiopaque barium multimer complexes (see, e.g., U.S. Pat. No.4,866,132), and the like.

A subject antibody will in some embodiments be linked to (e.g.,covalently or non-covalently linked) a fusion partner, e.g., a ligand;an epitope tag; a peptide; a protein other than an antibody; and thelike. Suitable fusion partners include peptides and polypeptides thatconfer enhanced stability in vivo (e.g., enhanced serum half-life);provide ease of purification, and the like; provide for secretion of thefusion protein from a cell; provide an epitope tag, e.g., (His)n, e.g.,6His, and the like; provide for secretion of the fusion protein from acell; provide an epitope tag, e.g., GST, hemagglutinin (HA; e.g.,CYPYDVPDYA; SEQ ID NO:10), FLAG (e.g., DYKDDDDK; SEQ ID NO:11), c-myc(e.g., CEQKLISEEDL; SEQ ID NO:12), and the like; provide a detectablesignal, e.g., an enzyme that generates a detectable product (e.g.,β-galactosidase, luciferase), or a protein that is itself detectable,e.g., a green fluorescent protein, a red fluorescent protein, a yellowfluorescent protein, etc.; provides for multimerization, e.g., amultimerization domain such as an Fc portion of an immunoglobulin; andthe like.

The fusion may also include an affinity domain, including peptidesequences that can interact with a binding partner, e.g., such as oneimmobilized on a solid support, useful for identification orpurification. Consecutive single amino acids, such as histidine, whenfused to a protein, can be used for one-step purification of the fusionprotein by high affinity binding to a resin column, such as nickelsepharose. Examples of affinity domains include His5 (HHHHH) (SEQ IDNO:13), HisX6 (HHHHHH) (SEQ ID NO:14), C-myc (EQKLISEEDL) (SEQ IDNO:15), Flag (DYKDDDDK) (SEQ ID NO:16), StrepTag (WSHPQFEK) (SEQ IDNO:17), hemagglutinin, e.g., HA Tag (YPYDVPDYA; SEQ ID NO:18),glutathinone-S-transferase (GST), thioredoxin, cellulose binding domain,RYIRS (SEQ ID NO:19), Phe-His-His-Thr (SEQ ID NO:20), chitin bindingdomain, S-peptide, T7 peptide, SH2 domain, C-end RNA tag,WEAAAREACCRECCARA (SEQ ID NO:21), metal binding domains, e.g., zincbinding domains or calcium binding domains such as those fromcalcium-binding proteins, e.g., calmodulin, troponin C, calcineurin B,myosin light chain, recoverin, S-modulin, visinin, VILIP, neurocalcin,hippocalcin, frequenin, caltractin, calpain large-subunit, S100proteins, parvalbumin, calbindin D9K, calbindin D28K, and calretinin,inteins, biotin, streptavidin, MyoD, leucine zipper sequences, andmaltose binding protein.

In some embodiments, a subject antibody comprises a polyaminemodification. A subject antibody can be modified with polyamines thatare either naturally occurring or synthetic. See, for example, U.S. Pat.No. 5,670,477. Useful naturally occurring polyamines include putrescine,spermidine, spermine, 1,3-deaminopropane, norspermidine,syn-homospermidine, thermine, thermospermine, caldopentamine,homocaldopentamine, and canavalmine. Putrescine, spermidine and spermineare particularly useful. Synthetic polyamines are composed of theempirical formula C_(X)H_(Y)N_(Z), can be cyclic or acyclic, branched orunbranched, hydrocarbon chains of 3-12 carbon atoms that further include1-6 NR or N(R)₂ moieties, wherein R is H, (C₁-C₄) alkyl, phenyl, orbenzyl. Polyamines can be linked to an antibody using any standardcrosslinking method.

In some embodiments, a subject antibody is modified to include acarbohydrate moiety, where the carbohydrate moiety can be covalentlylinked to the antibody. In some embodiments, a subject antibody ismodified to include a lipid moiety, where the lipid moiety can becovalently linked to the antibody. Suitable lipid moieties include,e.g., an N-fatty acyl group such as N-lauroyl, N-oleoyl, etc.; a fattyamine such as dodecyl amine, oleoyl amine, etc.; a C3-C16 long-chainaliphatic lipid; and the like. See, e.g., U.S. Pat. No. 6,638,513). Insome embodiments, a subject antibody is incorporated into a liposome.

Where an anti-CD22 antibody of the present disclosure comprises acovalently linked heterologous moiety, the heterologous moiety can belinked to the anti-CD22 heavy and/or light chain directly or via alinker. Suitable linkers can be readily selected and can be of any of asuitable of different lengths, such as from 1 amino acid (e.g., Gly) to20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acidsto 12 amino acids, including 4 amino acids to 10 amino acids, 5 aminoacids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acidsto 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

Examples of flexible linkers include glycine polymers (G)n,glycine-serine polymers (including, for example, (GS)n, GSGGSn (SEQ IDNO:22) and GGGSn (SEQ ID NO:23), where n is an integer of at least one),glycine-alanine polymers, alanine-serine polymers, and other flexiblelinkers known in the art. Glycine and glycine-serine polymers are ofinterest since both of these amino acids are relatively unstructured,and therefore may serve as a neutral tether between components. Glycinepolymers are of particular interest since glycine accesses significantlymore phi-psi space than even alanine, and is much less restricted thanresidues with longer side chains (see Scheraga, Rev. Computational Chem.11173-142 (1992)). Exemplary flexible linkers include, but are notlimited GGSG (SEQ ID NO:24), GGSGG (SEQ ID NO:25), GSGSG (SEQ ID NO:26), GSGGG (SEQ ID NO: 27), GGGSG (SEQ ID NO: 28), GSSSG (SEQ ID NO:29), and the like. The ordinarily skilled artisan will recognize thatdesign of a peptide conjugated to any elements described above caninclude linkers that are all or partially flexible, such that the linkercan include a flexible linker as well as one or more portions thatconfer less flexible structure.

Methods for Modification of Antibodies

The antibodies can be modified to have a covalently attachedheterologous moiety (e.g., detectable label, drug, etc.) by use of anyof a variety of methods. The present disclosure provides an anti-CD22antibody conjugated to a moiety of interest, where an anti-CD22 antibodyconjugated to a moiety of interest is referred to as an “anti-CD22antibody conjugate.” An anti-CD22 antibody conjugate of the presentdisclosure can include: 1) Ig heavy chain constant region conjugated toa moiety of interest; and an Ig light chain constant region conjugatedto a moiety of interest; 2) an Ig heavy chain constant region conjugatedto a moiety of interest; and an Ig light chain constant region that isnot conjugated to a moiety of interest; or 3) an Ig heavy chain constantregion that is not conjugated to a moiety of interest; and an Ig lightchain constant region conjugated to a moiety of interest. A subjectanti-CD22 antibody conjugate can also include VH and/or VL domains.

In one example, the antibody can be modified to include a2-formylglycine residue, which can serve as a chemical handle forattachment of a heterologous moiety. For example, the heavy and/or lightchain constant region of an anti-CD22 of the present disclosure can bemodified to include an amino acid sequence of a sulfatase motif which iscapable of being converted by action of a 2-formylglycine generatingenzyme (FGE) to contain a 2-formylglycine (FGly). Such sulfatase motifsmay also be referred to herein as an FGE-modification site. Action ofFGE is directed in a sequence-specific manner in that the FGE acts at asulfatase motif positioned within the immunoglobulin polypeptide. Themoiety of interest is provided as component of a reactive partner forreaction with an aldehyde of the FGly residue of a converted aldehydetag of the tagged Ig polypeptide. A wide range of commercially availablereagents can be used to accomplish attachment of a moiety of interest toan FGly residue of an aldehyde tagged Ig polypeptide. For example,aminooxy, hydrazide, or thiosemicarbazide derivatives of a number ofmoieties of interest are suitable reactive partners, and are readilyavailable or can be generated using standard chemical methods.

For example, to attach a poly(ethylene glycol) (PEG) moiety to a taggedIg polypeptide, an aminooxy-PEG can be generated from monoamino-PEGs andaminooxyglycine using standard protocols. The aminooxy-PEG can then bereacted with a converted (e.g., FGly-modified) aldehyde tagged Igpolypeptide to provide for attachment of the PEG moiety. Delivery of abiotin moiety to a converted aldehyde tagged polypeptide can beaccomplished using aminooxy biotin, biotin hydrazide or 2,4dinitrophenylhydrazine.

A minimal sulfatase motif of an aldehyde tag is usually 5 or 6 aminoacid residues in length, usually no more than 6 amino acid residues inlength. Sulfatase motifs provided in an Ig polypeptide are at least 5 or6 amino acid residues, and can be, for example, from 5 to 16, 6-16,5-15, 6-15, 5-14, 6-14, 5-13, 6-13, 5-12, 6-12, 5-11, 6-11, 5-10, 6-10,5-9, 6-9, 5-8, or 6-8 amino acid residues in length, so as to define asulfatase motif of less than 16, 15, 14, 13, 12, 11, 10, 9, 8 or 7 aminoacid residues in length. In certain embodiments, the sulfatase motifused may be described by the formula:X¹Z¹X²Z²X³Z³  (I)

where

Z¹ is cysteine or serine (which can also be represented by (C/S));

Z² is either a proline or alanine residue (which can also be representedby (P/A));

Z³ is a basic amino acid (e.g., arginine (R), and may be lysine (K) orhistidine (H), usually lysine), or an aliphatic amino acid (alanine (A),glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P),usually A, G, L, V, or I;

X¹ is present or absent and, when present, can be any amino acid, thoughusually an aliphatic amino acid, a sulfur-containing amino acid, or apolar, uncharged amino acid, (i.e., other than a aromatic amino acid ora charged amino acid), usually L, M, V, S or T, more usually L, M, S orV, with the proviso that when the sulfatase motif is at the N-terminusof the target polypeptide, X¹ is present; and

X² and X³ independently can be any amino acid, though usually analiphatic amino acid, a polar, uncharged amino acid, or a sulfurcontaining amino acid (i.e., other than a aromatic amino acid or acharged amino acid), e.g., S, T, A, V, G or C; e.g., S, T, A, V or G. Inone example, the aldehyde tag is of the formula L(C/S)TPSR (SEQ ID NO:30), e.g., LCTPSR (SEQ ID NO: 31) or LSTPSR (SEQ ID NO: 32). Thus, thepresent disclosure provides antibodies that include an aldehyde-taggedIg heavy chain and/or an aldehyde-tagged Ig light chain, where thealdehyde-tagged Ig antibody comprises an Ig constant region amino acidsequence of the heavy and/or light chain contains such a sulfatasemotif.

In general, the FGE used to facilitate conversion of cysteine or serineto FGly in a sulfatase motif of an aldehyde tag of a target polypeptideis selected according to the sulfatase motif present in the aldehydetag. The FGE can be native to the host cell in which the aldehyde taggedpolypeptide is expressed, or the host cell can be genetically modifiedto express an appropriate FGE. In some embodiments it may be desired touse a sulfatase motif compatible with a human FGE, and express thealdehyde tagged protein in a human cell that expresses the FGE or in ahost cell, usually a mammalian cell, genetically modified to express ahuman FGE. In general, an FGE suitable for use in generating anFGly-modified antibody can be obtained from naturally occurring sourcesor synthetically produced. For example, an appropriate FGE can bederived from biological sources which naturally produce an FGE or whichare genetically modified to express a recombinant gene encoding an FGE.Nucleic acids encoding a number of FGEs are known in the art andreadily.

Following action of an FGE on the sulfatase motif, Z₁ is oxidized togenerate a 2-formylglycine (FGly) residue. Furthermore, following bothFGE-mediated conversion and reaction with a reactive partner comprisinga moiety of interest, FGly position at Z₁ in the formula above iscovalently bound to the moiety of interest (e.g., detectable label,water soluble polymer, polypeptide, drug, etc.). Thus, the presentdisclosure provides an anti-CD22 antibody modified to comprise an FGlymoiety, wherein the anti-CD22 antibody comprises an FGly-convertedsulfatase motif of the formula:X¹(FGly)X²Z²X³Z³

wherein:

X¹ is present or absent and, when present, is any amino acid, with theproviso that when the sulfatase motif is at an N-terminus of thepolypeptide, X¹ is present;

X² and X³ are each independently any amino acid; and

Z³ is a basic amino acid; and

where the FGly-modified anti-CD22 antibody presents the FGly group on asolvent-accessible surface when in a folded state. In some embodiments,the FGly-converted sulfatase motif is of the formula L(FGly)TPSR (SEQ IDNO: 33).

As noted above, a subject anti-CD22 antibody modified to include an FGlymoiety can be further modified to include a heterologous moiety ofinterest (e.g., detectable label, water soluble polymer, polypeptide,drug, etc.) covalently bound to the anti-CD22 antibody via the FGlymoiety. Thus, the present disclosure provides an anti-CD22 antibodyconjugate (also referred to herein as an “anti-CD22 conjugate”), theanti-CD22 conjugate comprising:X¹(FGly′)X²Z²X³Z³  (I′)

where

FGly′ is the 2-formylglycine residue having a covalently attachedmoiety;

Z² is either a proline or alanine residue (which can also be representedby (P/A)); Z³ is a basic amino acid (e.g., arginine (R), and may belysine (K) or histidine (H), usually lysine), or an aliphatic amino acid(alanine (A), glycine (G), leucine (L), valine (V), isoleucine (I), orproline (P), usually A, G, L, V, or I;

X¹ may be present or absent and, when present, can be any amino acid,though usually an aliphatic amino acid, a sulfur-containing amino acid,or a polar, uncharged amino acid, (i.e., other than a aromatic aminoacid or a charged amino acid), usually L, M, V, S or T, more usually L,M or V, with the proviso that when the sulfatase motif is at theN-terminus of the target polypeptide, X¹ is present; and

X² and X³ independently can be any amino acid, though usually analiphatic amino acid, a sulfur-containing amino acid, or a polar,uncharged amino acid, (i.e., other than a aromatic amino acid or acharged amino acid), usually S, T, A, V, G or C, more usually S, T, A, Vor G. In some embodiments, the motif is of the formula L(FGly′)TPSR (SEQID NO: 34).

Drugs

In some cases, an anti-CD22 antibody of the present disclosure comprisesdrug covalently linked to the heavy and/or light chain of the antibody.“Drugs” include small molecule drugs, peptidic drugs, toxins (e.g.,cytotoxins), and the like.

“Small molecule drug” as used herein refers to a compound, e.g., anorganic compound, which exhibits a pharmaceutical activity of interestand which is generally of a molecular weight of no greater than about800 Da, or no greater than 2000 Da, but can encompass molecules of up to5 kDa and can be as large as about 10 kDa. A small inorganic moleculerefers to a molecule containing no carbon atoms, while a small organicmolecule refers to a compound containing at least one carbon atom.

“Peptide drug” as used herein refers to amino-acid containing polymericcompounds, and is meant to encompass naturally-occurring andnon-naturally-occurring peptides, oligopeptides, cyclic peptides,polypeptides, and proteins, as well as peptide mimetics. The peptidedrugs may be obtained by chemical synthesis or be produced from agenetically encoded source (e.g., recombinant source). Peptide drugs canrange in molecular weight, and can be from 200 Da to 10 kDa or greaterin molecular weight.

In some cases, the drug is a toxin, e.g., a cytotoxin. Ribosomeinactivating proteins (RIPs), which are a class of proteins ubiquitousin higher plants, are examples of such cytotoxins. RIPs, which aredivided into Type I and Type II classes, are cytotoxic due to theiractivity as potent inhibitors of eukaryotic protein synthesis. Type IRIPS are composed of a single peptide chain having ribosome-inactivatingactivity, while Type II proteins are composed of an A-chain, essentiallyequivalent to a Type I protein, disulfide-linked to a B-chain havingcell-binding properties. The N-glycosidic bond of a specific adeninebase is hydrolytically cleaved by RIPs in a highly conserved loop regionof the 28S rRNA of eukaryotic ribosomes, thereby inactivatingtranslation in eukaryotic cells. See, e.g., U.S. Pat. No. 5,744,580.Gelonin, dodecandrin, tricosanthin, tricokirin, bryodin, Mirabilisantiviral protein (MAP), barley ribosome-inactivating protein (BRIP),pokeweed antiviral proteins (PAPS), saporins, luffins, and momordins areexamples of Type I RIPs; whereas ricin and abrin are examples of Type IIRIPS. Suitable cytotoxins include, but are not limited to, ricin, abrin,diphtheria toxin, a Pseudomonas exotoxin (e.g., PE35, PE37, PE38, PE40,etc.), saporin, gelonin, a pokeweed anti-viral protein (PAP), botulinumtoxin, bryodin, momordin, and bouganin.

In some cases, the drug is a cancer chemotherapeutic agent. Cancerchemotherapeutic agents include non-peptidic (i.e., non-proteinaceous)compounds that reduce proliferation of cancer cells, and encompasscytotoxic agents and cytostatic agents. Non-limiting examples ofchemotherapeutic agents include alkylating agents, nitrosoureas,antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, andsteroid hormones. Peptidic compounds can also be used. Suitable cancerchemotherapeutic agents include dolastatin and active analogs andderivatives thereof; and auristatin and active analogs and derivativesthereof. See, e.g., WO 96/33212, WO 96/14856, and U.S. Pat. No.6,323,315. For example, dolastatin 10 or auristatin PE can be includedin an antibody-drug conjugate of the present disclosure. Suitable cancerchemotherapeutic agents also include maytansinoids and active analogsand derivatives thereof (see, e.g., EP 1391213; and Liu et al (1996)Proc. Natl. Acad. Sci. USA 93:8618-8623); and duocarmycins and activeanalogs and derivatives thereof (e.g., including the syntheticanalogues, KW-2189 and CB 1-TM1).

Agents that act to reduce cellular proliferation are known in the artand widely used. Such agents include alkylating agents, such as nitrogenmustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates, andtriazenes, including, but not limited to, mechlorethamine,cyclophosphamide (Cytoxan™), melphalan (L-sarcolysin), carmustine(BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin,chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil,pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan,dacarbazine, and temozolomide.

Antimetabolite agents include folic acid analogs, pyrimidine analogs,purine analogs, and adenosine deaminase inhibitors, including, but notlimited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil(5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP),pentostatin, 5-fluorouracil (5-FU), methotrexate,10-propargyl-5,8-dideazafolate (PDDF, CB3717),5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabinephosphate, pentostatine, and gemcitabine.

Suitable natural products and their derivatives, (e.g., vinca alkaloids,antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins),include, but are not limited to, Ara-C, paclitaxel (Taxol®), docetaxel(Taxotere®), deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine;brequinar; alkaloids, e.g. vincristine, vinblastine, vinorelbine,vindesine, etc.; podophyllotoxins, e.g. etoposide, teniposide, etc.;antibiotics, e.g. anthracycline, daunorubicin hydrochloride (daunomycin,rubidomycin, cerubidine), idarubicin, doxorubicin, epirubicin andmorpholino derivatives, etc.; phenoxizone biscyclopeptides, e.g.dactinomycin; basic glycopeptides, e.g. bleomycin; anthraquinoneglycosides, e.g. plicamycin (mithramycin); anthracenediones, e.g.mitoxantrone; azirinopyrrolo indolediones, e.g. mitomycin; macrocyclicimmunosuppressants, e.g. cyclosporine, FK-506 (tacrolimus, prograf),rapamycin, etc.; and the like.

Other anti-proliferative cytotoxic agents are navelbene, CPT-11,anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide,ifosamide, and droloxafine.

Microtubule affecting agents that have antiproliferative activity arealso suitable for use and include, but are not limited to,allocolchicine (NSC 406042), Halichondrin B (NSC 609395), colchicine(NSC 757), colchicine derivatives (e.g., NSC 33410), dolstatin 10 (NSC376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel(Taxol®), Taxol® derivatives, docetaxel (Taxotere®), thiocolchicine (NSC361792), trityl cysterin, vinblastine sulfate, vincristine sulfate,natural and synthetic epothilones including but not limited to,eopthilone A, epothilone B, discodermolide; estramustine, nocodazole,and the like.

Hormone modulators and steroids (including synthetic analogs) that aresuitable for use include, but are not limited to, adrenocorticosteroids,e.g. prednisone, dexamethasone, etc.; estrogens and pregestins, e.g.hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrolacetate, estradiol, clomiphene, tamoxifen; etc.; and adrenocorticalsuppressants, e.g. aminoglutethimide; 17α-ethinylestradiol;diethylstilbestrol, testosterone, fluoxymesterone, dromostanolonepropionate, testolactone, methylprednisolone, methyl-testosterone,prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone,aminoglutethimide, estramustine, medroxyprogesterone acetate,leuprolide, Flutamide (Drogenil), Toremifene (Fareston), and Zoladex®.Estrogens stimulate proliferation and differentiation; thereforecompounds that bind to the estrogen receptor are used to block thisactivity.

Other suitable chemotherapeutic agents include metal complexes, e.g.cisplatin (cis-DDP), carboplatin, etc.; ureas, e.g. hydroxyurea; andhydrazines, e.g. N-methylhydrazine; epidophyllotoxin; a topoisomeraseinhibitor; procarbazine; mitoxantrone; leucovorin; tegafur; etc. Otheranti-proliferative agents of interest include immunosuppressants, e.g.mycophenolic acid, thalidomide, desoxyspergualin, azasporine,leflunomide, mizoribine, azaspirane (SKF 105685); Iressa® (ZD 1839,4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline);etc.

Taxanes are suitable for use. “Taxanes” include paclitaxel, as well asany active taxane derivative or pro-drug. “Paclitaxel” (which should beunderstood herein to include analogues, formulations, and derivativessuch as, for example, docetaxel, TAXOL□, TAXOTERE□ docetaxel),10-desacetyl analogs of paclitaxel and3′N-desbenzoyl-3′N-t-butoxycarbonyl analogs of paclitaxel) may bereadily prepared utilizing techniques known to those skilled in the art(see also WO 94/07882, WO 94/07881, WO 94/07880, WO 94/07876, WO93/23555, WO 93/10076; U.S. Pat. Nos. 5,294,637; 5,283,253; 5,279,949;5,274,137; 5,202,448; 5,200,534; 5,229,529; and EP 590,267), or obtainedfrom a variety of commercial sources, including for example, SigmaChemical Co., St. Louis, Mo. (T7402 from Taxus brevifolia; or T-1912from Taxus yannanensis).

Paclitaxel should be understood to refer to not only the commonchemically available form of paclitaxel, but analogs and derivatives(e.g., Taxotere□ docetaxel, a conjugates (e.g., paclitaxel-PEG,paclitaxel-dextran, or paclitaxel-xylose).

Also included within the term “taxane” are a variety of knownderivatives, including both hydrophilic derivatives, and hydrophobicderivatives. Taxane derivatives include, but not limited to, galactoseand mannose derivatives described in International Patent ApplicationNo. WO 99/18113; piperazino and other derivatives described in WO99/14209; taxane derivatives described in WO 99/09021, WO 98/22451, andU.S. Pat. No. 5,869,680; 6-thio derivatives described in WO 98/28288;sulfenamide derivatives described in U.S. Pat. No. 5,821,263; and taxolderivative described in U.S. Pat. No. 5,415,869. It further includesprodrugs of paclitaxel including, but not limited to, those described inWO 98/58927; WO 98/13059; and U.S. Pat. No. 5,824,701.

Methods of Producing Antibody

A subject antibody can be produced by any known method, e.g.,conventional synthetic methods for protein synthesis; recombinant DNAmethods; etc.

Where a subject antibody is a single chain polypeptide, it can besynthesized using standard chemical peptide synthesis techniques. Wherea polypeptide is chemically synthesized, the synthesis may proceed vialiquid-phase or solid-phase. Solid phase polypeptide synthesis (SPPS),in which the C-terminal amino acid of the sequence is attached to aninsoluble support followed by sequential addition of the remaining aminoacids in the sequence, is an example of a suitable method for thechemical synthesis of a subject antibody. Various forms of SPPS, such asFmoc and Boc, are available for synthesizing a subject antibody.Techniques for solid phase synthesis are described by Barany andMerrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides:Analysis, Synthesis, Biology. Vol. 2: Special Methods in PeptideSynthesis, Part A., Merrifield, et al. J. Am. Chem. Soc., 85: 2149-2156(1963); Stewart et al., Solid Phase Peptide Synthesis, 2nd ed. PierceChem. Co., Rockford, Ill. (1984); and Ganesan A. 2006 Mini Rev. MedChem. 6:3-10 and Camarero J A et al. 2005 Protein Pept Lett. 12:723-8.Briefly, small insoluble, porous beads are treated with functional unitson which peptide chains are built. After repeated cycling ofcoupling/deprotection, the free N-terminal amine of a solid-phaseattached is coupled to a single N-protected amino acid unit. This unitis then deprotected, revealing a new N-terminal amine to which a furtheramino acid may be attached. The peptide remains immobilized on thesolid-phase and undergoes a filtration process before being cleaved off.Standard recombinant methods can be used for production of a subjectantibody. For example, nucleic acids encoding light and heavy chainvariable regions, optionally linked to constant regions, are insertedinto expression vectors. The light and heavy chains can be cloned in thesame or different expression vectors. The DNA segments encodingimmunoglobulin chains are operably linked to control sequences in theexpression vector(s) that ensure the expression of immunoglobulinpolypeptides. Expression control sequences include, but are not limitedto, promoters (e.g., naturally-associated or heterologous promoters),signal sequences, enhancer elements, and transcription terminationsequences. The expression control sequences can be eukaryotic promotersystems in vectors capable of transforming or transfecting eukaryotichost cells (e.g., COS or CHO cells). Once the vector has beenincorporated into the appropriate host, the host is maintained underconditions suitable for high level expression of the nucleotidesequences, and the collection and purification of the antibodies.

Because of the degeneracy of the code, a variety of nucleic acidsequences can encode each immunoglobulin amino acid sequence. Thedesired nucleic acid sequences can be produced by de novo solid-phaseDNA synthesis or by polymerase chain reaction (PCR) mutagenesis of anearlier prepared variant of the desired polynucleotide.Oligonucleotide-mediated mutagenesis is an example of a suitable methodfor preparing substitution, deletion and insertion variants of targetpolypeptide DNA. See Adelman et al., DNA 2:183 (1983). Briefly, thetarget polypeptide DNA is altered by hybridizing an oligonucleotideencoding the desired mutation to a single-stranded DNA template. Afterhybridization, a DNA polymerase is used to synthesize an entire secondcomplementary strand of the template that incorporates theoligonucleotide primer, and encodes the selected alteration in thetarget polypeptide DNA.

Suitable expression vectors are typically replicable in the hostorganisms either as episomes or as an integral part of the hostchromosomal DNA. Commonly, expression vectors contain selection markers(e.g., ampicillin-resistance, hygromycin-resistance, tetracyclineresistance, kanamycin resistance or neomycin resistance) to permitdetection of those cells transformed with the desired DNA sequences.

Escherichia coli is an example of a prokaryotic host cell that can beused for cloning a subject antibody-encoding polynucleotide. Othermicrobial hosts suitable for use include bacilli, such as Bacillussubtilis, and other enterobacteriaceae, such as Salmonella, Serratia,and various Pseudomonas species. In these prokaryotic hosts, one canalso make expression vectors, which will typically contain expressioncontrol sequences compatible with the host cell (e.g., an origin ofreplication). In addition, any number of a variety of well-knownpromoters will be present, such as the lactose promoter system, atryptophan (trp) promoter system, a beta-lactamase promoter system, or apromoter system from phage lambda. The promoters will typically controlexpression, optionally with an operator sequence, and have ribosomebinding site sequences and the like, for initiating and completingtranscription and translation.

Other microbes, such as yeast, are also useful for expression.Saccharomyces (e.g., S. cerevisiae) and Pichia are examples of suitableyeast host cells, with suitable vectors having expression controlsequences (e.g., promoters), an origin of replication, terminationsequences and the like as desired. Typical promoters include3-phosphoglycerate kinase and other glycolytic enzymes.

Inducible yeast promoters include, among others, promoters from alcoholdehydrogenase, isocytochrome C, and enzymes responsible for maltose andgalactose utilization.

In addition to microorganisms, mammalian cells (e.g., mammalian cellsgrown in in vitro cell culture) can also be used to express and producethe polypeptides of the present invention (e.g., polynucleotidesencoding immunoglobulins or fragments thereof). See Winnacker, FromGenes to Clones, VCH Publishers, N.Y., N.Y. (1987). Suitable mammalianhost cells include CHO cell lines, various Cos cell lines, HeLa cells,myeloma cell lines, and transformed B-cells or hybridomas. Expressionvectors for these cells can include expression control sequences, suchas an origin of replication, a promoter, and an enhancer (Queen et al.,Immunol. Rev. 89:49 (1986)), and necessary processing information sites,such as ribosome binding sites, RNA splice sites, polyadenylation sites,and transcriptional terminator sequences. Examples of suitableexpression control sequences are promoters derived from immunoglobulingenes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus and thelike. See Co et al., J. Immunol. 148:1149 (1992).

Once synthesized (either chemically or recombinantly), the wholeantibodies, their dimers, individual light and heavy chains, or otherforms of a subject antibody (e.g., scFv, etc.) can be purified accordingto standard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, high performanceliquid chromatography (HPLC) purification, gel electrophoresis, and thelike (see generally Scopes, Protein Purification (Springer-Verlag, N.Y.,(1982)). A subject antibody can be substantially pure, e.g., at leastabout 80% to 85% pure, at least about 85% to 90% pure, at least about90% to 95% pure, or 98% to 99%, or more, pure, e.g., free fromcontaminants such as cell debris, macromolecules other than a subjectantibody, etc.

Compositions

The present disclosure provides a composition comprising a subjectantibody. A subject antibody composition can comprise, in addition to asubject antibody, one or more of: a salt, e.g., NaCl, MgCl₂, KCl, MgSO₄,etc.; a buffering agent, e.g., a Tris buffer,N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),2-(N-Morpholino)ethanesulfonic acid (MES),2-(N-Morpholino)ethanesulfonic acid sodium salt (MES),3-(N-Morpholino)propanesulfonic acid (MOPS),N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; asolubilizing agent; a detergent, e.g., a non-ionic detergent such asTween-20, etc.; a protease inhibitor; glycerol; and the like.

Nucleic Acids

The present disclosure provides nucleic acids comprising nucleotidesequences encoding a subject antibody. A nucleotide sequence encoding asubject antibody can be operably linked to one or more regulatoryelements, such as a promoter and enhancer, that allow expression of thenucleotide sequence in the intended target cells (e.g., a cell that isgenetically modified to synthesize the encoded antibody).

Suitable promoter and enhancer elements are known in the art. Forexpression in a bacterial cell, suitable promoters include, but are notlimited to, lad, lacZ, T3, T7, gpt, lambda P and trc. For expression ina eukaryotic cell, suitable promoters include, but are not limited to,light and/or heavy chain immunoglobulin gene promoter and enhancerelements; cytomegalovirus immediate early promoter; herpes simplex virusthymidine kinase promoter; early and late SV40 promoters; promoterpresent in long terminal repeats from a retrovirus; mousemetallothionein-I promoter; and various art-known tissue specificpromoters.

In some embodiments, e.g., for expression in a yeast cell, a suitablepromoter is a constitutive promoter such as an ADH1 promoter, a PGK1promoter, an ENO promoter, a PYK1 promoter and the like; or aregulatable promoter such as a GAL1 promoter, a GAL10 promoter, an ADH2promoter, a PHO5 promoter, a CUP1 promoter, a GAL7 promoter, a MET25promoter, a MET3 promoter, a CYC1 promoter, a HIS3 promoter, an ADH1promoter, a PGK promoter, a GAPDH promoter, an ADC1 promoter, a TRP1promoter, a URA3 promoter, a LEU2 promoter, an ENO promoter, a TP1promoter, and AOX1 (e.g., for use in Pichia). Selection of theappropriate vector and promoter is well within the level of ordinaryskill in the art.

Suitable promoters for use in prokaryotic host cells include, but arenot limited to, a bacteriophage T7 RNA polymerase promoter; a trppromoter; a lac operon promoter; a hybrid promoter, e.g., a lac/tachybrid promoter, a tac/trc hybrid promoter, a trp/lac promoter, a T7/lacpromoter; a trc promoter; a tac promoter, and the like; an araBADpromoter; in vivo regulated promoters, such as an ssaG promoter or arelated promoter (see, e.g., U.S. Patent Publication No. 20040131637), apagC promoter (Pulkkinen and Miller, J. Bacteriol., 1991: 173(1): 86-93;Alpuche-Aranda et al., PNAS, 1992; 89(21): 10079-83), a nirB promoter(Harborne et al. (1992) Mol. Micro. 6:2805-2813), and the like (see,e.g., Dunstan et al. (1999) Infect. Immun. 67:5133-5141; McKelvie et al.(2004) Vaccine 22:3243-3255; and Chatfield et al. (1992) Biotechnol.10:888-892); a sigma70 promoter, e.g., a consensus sigma70 promoter(see, e.g., GenBank Accession Nos. AX798980, AX798961, and AX798183); astationary phase promoter, e.g., a dps promoter, an spy promoter, andthe like; a promoter derived from the pathogenicity island SPI-2 (see,e.g., WO96/17951); an actA promoter (see, e.g., Shetron-Rama et al.(2002) Infect. Immun. 70:1087-1096); an rpsM promoter (see, e.g.,Valdivia and Falkow (1996). Mol. Microbiol. 22:367); a tet promoter(see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. andHeinemann, U. (eds), Topics in Molecular and Structural Biology,Protein-Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp.143-162); an SP6 promoter (see, e.g., Melton et al. (1984) Nucl. AcidsRes. 12:7035); and the like. Suitable strong promoters for use inprokaryotes such as Escherichia coli include, but are not limited toTrc, Tac, T5, T7, and P_(Lambda.) Non-limiting examples of operators foruse in bacterial host cells include a lactose promoter operator (LacIrepressor protein changes conformation when contacted with lactose,thereby preventing the LacI repressor protein from binding to theoperator), a tryptophan promoter operator (when complexed withtryptophan, TrpR repressor protein has a conformation that binds theoperator; in the absence of tryptophan, the TrpR repressor protein has aconformation that does not bind to the operator), and a tac promoteroperator (see, for example, deBoer et al. (1983) Proc. Natl. Acad. Sci.U.S.A. 80:21-25).

A nucleotide sequence encoding a subject antibody can be present in anexpression vector and/or a cloning vector. Where a subject antibodycomprises two separate polypeptides, nucleotide sequences encoding thetwo polypeptides can be cloned in the same or separate vectors. Anexpression vector can include a selectable marker, an origin ofreplication, and other features that provide for replication and/ormaintenance of the vector.

Large numbers of suitable vectors and promoters are known to those ofskill in the art; many are commercially available for generating asubject recombinant constructs. The following vectors are provided byway of example. Bacterial: pBs, phagescript, PsiX174, pBluescript SK,pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif,USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia,Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG(Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences encoding heterologous proteins. A selectable marker operativein the expression host may be present. Suitable expression vectorsinclude, but are not limited to, viral vectors (e.g. viral vectors basedon vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., InvestOpthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., HGene Ther 5:1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (see,e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:28572863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al.,Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594,1996; Srivastava in WO 93/09239, Samulski et al., J. Vir. (1989)63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte etal., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; humanimmunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:10319 23,1997; Takahashi et al., J Virol 73:7812 7816, 1999); a retroviral vector(e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derivedfrom retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus,avian leukosis virus, human immunodeficiency virus, myeloproliferativesarcoma virus, and mammary tumor virus); and the like.

As noted above, a subject nucleic acid comprises a nucleotide sequenceencoding a subject antibody. A subject nucleic acid can comprise anucleotide sequence encoding anti-CD22 heavy- and light-chains, asdescribed above.

Cells

The present disclosure provides isolated genetically modified host cells(e.g., in vitro cells) that are genetically modified with a subjectnucleic acid. In some embodiments, a subject isolated geneticallymodified host cell can produce a subject antibody.

Suitable host cells include eukaryotic host cells, such as a mammaliancell, an insect host cell, a yeast cell; and prokaryotic cells, such asa bacterial cell. Introduction of a subject nucleic acid into the hostcell can be effected, for example by calcium phosphate precipitation,DEAE dextran mediated transfection, liposome-mediated transfection,electroporation, or other known method. Suitable mammalian cells includeprimary cells and immortalized cell lines. Suitable mammalian cell linesinclude human cell lines, non-human primate cell lines, rodent (e.g.,mouse, rat) cell lines, and the like. Suitable mammalian cell linesinclude, but are not limited to, HeLa cells (e.g., American Type CultureCollection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61,CRL9096), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721),COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells(ATCC No. CCLI.3), human embryonic kidney (HEK) 293 cells (ATCC No.CRL1573), HLHepG2 cells, and the like. Suitable yeast cells include, butare not limited to, Pichia pastoris, Pichia finlandica, Pichiatrehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae,Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichiapijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomycescerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp.,Kluyveromyces lactis, Candida albicans, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporiumlucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum,Neurospora crassa, Chlamydomonas reinhardtii, and the like.

Suitable prokaryotic cells include, but are not limited to, any of avariety of laboratory strains of Escherichia coli, Lactobacillus sp.,Salmonella sp., Shigella sp., and the like. See, e.g., Carrier et al.(1992) J. Immunol. 148:1176-1181; U.S. Pat. No. 6,447,784; and Sizemoreet al. (1995) Science 270:299-302. Examples of Salmonella strains whichcan be employed in the present invention include, but are not limitedto, Salmonella typhi and S. typhimurium. Suitable Shigella strainsinclude, but are not limited to, Shigella flexneri, Shigella sonnei, andShigella disenteriae. Typically, the laboratory strain is one that isnon-pathogenic. Non-limiting examples of other suitable bacteriainclude, but are not limited to, Bacillus subtilis, Pseudomonas pudita,Pseudomonas aeruginosa, Pseudomonas mevalonii, Rhodobacter sphaeroides,Rhodobacter capsulatus, Rhodospirillum rubrum, Rhodococcus sp., and thelike. In some embodiments, the host cell is Escherichia coli.

Pharmaceutical Compositions

The present disclosure provides compositions, including pharmaceuticalcompositions, comprising a subject antibody. In general, a formulationcomprises an effective amount of a subject antibody. An “effectiveamount” means a dosage sufficient to produce a desired result, e.g.,reduction in the number of cancerous B cells, reduction in the numberand/or activity of autoreactive B cells. In some cases, the desiredresult is at least a reduction in a symptom of a B cell malignancy, ascompared to a control.

Formulations

In the subject methods, a subject antibody can be administered to thehost using any convenient means capable of resulting in the desiredtherapeutic effect or diagnostic effect. Thus, the agent can beincorporated into a variety of formulations for therapeuticadministration. More particularly, a subject antibody can be formulatedinto pharmaceutical compositions by combination with appropriate,pharmaceutically acceptable carriers or diluents, and may be formulatedinto preparations in solid, semi-solid, liquid or gaseous forms, such astablets, capsules, powders, granules, ointments, solutions,suppositories, injections, inhalants and aerosols.

In pharmaceutical dosage forms, a subject antibody can be administeredin the form of their pharmaceutically acceptable salts, or they may alsobe used alone or in appropriate association, as well as in combination,with other pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

For oral preparations, a subject antibody can be used alone or incombination with appropriate additives to make tablets, powders,granules or capsules, for example, with conventional additives, such aslactose, mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

A subject antibody can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

Pharmaceutical compositions comprising a subject antibody are preparedby mixing the antibody having the desired degree of purity with optionalphysiologically acceptable carriers, excipients, stabilizers,surfactants, buffers and/or tonicity agents. Acceptable carriers,excipients and/or stabilizers are nontoxic to recipients at the dosagesand concentrations employed, and include buffers such as phosphate,citrate, and other organic acids; antioxidants including ascorbic acid,glutathione, cysteine, methionine and citric acid; preservatives (suchas ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methylor propyl parabens, benzalkonium chloride, or combinations thereof);amino acids such as arginine, glycine, ornithine, lysine, histidine,glutamic acid, aspartic acid, isoleucine, leucine, alanine,phenylalanine, tyrosine, tryptophan, methionine, serine, proline andcombinations thereof; monosaccharides, disaccharides and othercarbohydrates; low molecular weight (less than about 10 residues)polypeptides; proteins, such as gelatin or serum albumin; chelatingagents such as EDTA; sugars such as trehalose, sucrose, lactose,glucose, mannose, maltose, galactose, fructose, sorbose, raffinose,glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid;and/or non-ionic surfactants such as Tween, Brij Pluronics, Triton-X, orpolyethylene glycol (PEG).

The pharmaceutical composition may be in a liquid form, a lyophilizedform or a liquid form reconstituted from a lyophilized form, wherein thelyophilized preparation is to be reconstituted with a sterile solutionprior to administration. The standard procedure for reconstituting alyophilized composition is to add back a volume of pure water (typicallyequivalent to the volume removed during lyophilization); howeversolutions comprising antibacterial agents may be used for the productionof pharmaceutical compositions for parenteral administration; see alsoChen (1992) Drug Dev Ind Pharm 18, 1311-54.

Exemplary antibody concentrations in a subject pharmaceuticalcomposition may range from about 1 mg/mL to about 200 mg/ml or fromabout 50 mg/mL to about 200 mg/mL, or from about 150 mg/mL to about 200mg/mL.

An aqueous formulation of the antibody may be prepared in a pH-bufferedsolution, e.g., at pH ranging from about 4.0 to about 7.0, or from about5.0 to about 6.0, or alternatively about 5.5. Examples of buffers thatare suitable for a pH within this range include phosphate-, histidine-,citrate-, succinate-, acetate-buffers and other organic acid buffers.The buffer concentration can be from about 1 mM to about 100 mM, or fromabout 5 mM to about 50 mM, depending, e.g., on the buffer and thedesired tonicity of the formulation.

A tonicity agent may be included in the antibody formulation to modulatethe tonicity of the formulation. Exemplary tonicity agents includesodium chloride, potassium chloride, glycerin and any component from thegroup of amino acids, sugars as well as combinations thereof. In someembodiments, the aqueous formulation is isotonic, although hypertonic orhypotonic solutions may be suitable. The term “isotonic” denotes asolution having the same tonicity as some other solution with which itis compared, such as physiological salt solution or serum. Tonicityagents may be used in an amount of about 5 mM to about 350 mM, e.g., inan amount of 100 mM to 350 nM.

A surfactant may also be added to the antibody formulation to reduceaggregation of the formulated antibody and/or minimize the formation ofparticulates in the formulation and/or reduce adsorption. Exemplarysurfactants include polyoxyethylensorbitan fatty acid esters (Tween),polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers(Triton-X), polyoxyethylene-polyoxypropylene copolymer (Poloxamer,Pluronic), and sodium dodecyl sulfate (SDS). Examples of suitablepolyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (soldunder the trademark Tween 20™) and polysorbate 80 (sold under thetrademark Tween 80™). Examples of suitable polyethylene-polypropylenecopolymers are those sold under the names Pluronic® F68 or Poloxamer188™. Examples of suitable Polyoxyethylene alkyl ethers are those soldunder the trademark Brij™. Exemplary concentrations of surfactant mayrange from about 0.001% to about 1% w/v.

A lyoprotectant may also be added in order to protect the labile activeingredient (e.g. a protein) against destabilizing conditions during thelyophilization process. For example, known lyoprotectants include sugars(including glucose and sucrose); polyols (including mannitol, sorbitoland glycerol); and amino acids (including alanine, glycine and glutamicacid). Lyoprotectants can be included in an amount of about 10 mM to 500nM.

In some embodiments, a subject formulation includes a subject antibody,and one or more of the above-identified agents (e.g., a surfactant, abuffer, a stabilizer, a tonicity agent) and is essentially free of oneor more preservatives, such as ethanol, benzyl alcohol, phenol,m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkoniumchloride, and combinations thereof. In other embodiments, a preservativeis included in the formulation, e.g., at concentrations ranging fromabout 0.001 to about 2% (w/v).

For example, a subject formulation can be a liquid or lyophilizedformulation suitable for parenteral administration, and can comprise:about 1 mg/mL to about 200 mg/mL of a subject antibody; about 0.001% toabout 1% of at least one surfactant; about 1 mM to about 100 mM of abuffer; optionally about 10 mM to about 500 mM of a stabilizer; andabout 5 mM to about 305 mM of a tonicity agent; and has a pH of about4.0 to about 7.0.

As another example, a subject parenteral formulation is a liquid orlyophilized formulation comprising: about 1 mg/mL to about 200 mg/mL ofa subject antibody; 0.04% Tween 20 w/v; 20 mM L-histidine; and 250 mMSucrose; and has a pH of 5.5.

As another example, a subject parenteral formulation comprises alyophilized formulation comprising: 1) 15 mg/mL of a subject antibody;0.04% Tween 20 w/v; 20 mM L-histidine; and 250 mM sucrose; and has a pHof 5.5; or 2) 75 mg/mL of a subject antibody; 0.04% Tween 20 w/v; 20 mML-histidine; and 250 mM sucrose; and has a pH of 5.5; or 3) 75 mg/mL ofa subject antibody; 0.02% Tween 20 w/v; 20 mM L-histidine; and 250 mMSucrose; and has a pH of 5.5; or 4) 75 mg/mL of a subject antibody;0.04% Tween 20 w/v; 20 mM L-histidine; and 250 mM trehalose; and has apH of 5.5; or 6) 75 mg/mL of a subject antibody; 0.02% Tween 20 w/v; 20mM L-histidine; and 250 mM trehalose; and has a pH of 5.5.

As another example, a subject parenteral formulation is a liquidformulation comprising: 1) 7.5 mg/mL of a subject antibody; 0.022% Tween20 w/v; 120 mM L-histidine; and 250 125 mM sucrose; and has a pH of 5.5;or 2) 37.5 mg/mL of a subject antibody; 0.02% Tween 20 w/v; 10 mML-histidine; and 125 mM sucrose; and has a pH of 5.5; or 3) 37.5 mg/mLof a subject antibody; 0.01% Tween 20 w/v; 10 mM L-histidine; and 125 mMsucrose; and has a pH of 5.5; or 4) 37.5 mg/mL of a subject antibody;0.02% Tween 20 w/v; 10 mM L-histidine; 125 mM trehalose; and has a pH of5.5; or 5) 37.5 mg/mL of a subject antibody; 0.01% Tween 20 w/v; 10 mML-histidine; and 125 mM trehalose; and has a pH of 5.5; or 6) 5 mg/mL ofa subject antibody; 0.02% Tween 20 w/v; 20 mM L-histidine; and 250 mMtrehalose; and has a pH of 5.5; or 7) 75 mg/mL of a subject antibody;0.02% Tween 20 w/v; 20 mM L-histidine; and 250 mM mannitol; and has a pHof 5.5; or 8) 75 mg/mL of a subject antibody; 0.02% Tween 20 w/v; 20 mML histidine; and 140 mM sodium chloride; and has a pH of 5.5; or 9) 150mg/mL of a subject antibody; 0.02% Tween 20 w/v; 20 mM L-histidine; and250 mM trehalose; and has a pH of 5.5; or 10) 150 mg/mL of a subjectantibody; 0.02% Tween 20 w/v; 20 mM L-histidine; and 250 mM mannitol;and has a pH of 5.5; or 11) 150 mg/mL of a subject antibody; 0.02% Tween20 w/v; 20 mM L-histidine; and 140 mM sodium chloride; and has a pH of5.5; or 12) 10 mg/mL of a subject antibody; 0.01% Tween 20 w/v; 20 mML-histidine; and 40 mM sodium chloride; and has a pH of 5.5.

A subject antibody can be utilized in aerosol formulation to beadministered via inhalation. A subject antibody can be formulated intopressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, a subject antibody can be made into suppositories by mixingwith a variety of bases such as emulsifying bases or water-solublebases. A subject antibody can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or moreinhibitors. Similarly, unit dosage forms for injection or intravenousadministration may comprise a subject antibody in a composition as asolution in sterile water, normal saline or another pharmaceuticallyacceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for a subject antibodymay depend on the particular antibody employed and the effect to beachieved, and the pharmacodynamics associated with each antibody in thehost. Other modes of administration will also find use with the subjectinvention. For instance, a subject antibody can be formulated insuppositories and, in some cases, aerosol and intranasal compositions.For suppositories, the vehicle composition will include traditionalbinders and carriers such as, polyalkylene glycols, or triglycerides.Such suppositories may be formed from mixtures containing the activeingredient in the range of about 0.5% to about 10% (w/w), e.g., about 1%to about 2%.

Intranasal formulations will usually include vehicles that neither causeirritation to the nasal mucosa nor significantly disturb ciliaryfunction. Diluents such as water, aqueous saline or other knownsubstances can be employed with the subject invention. The nasalformulations may also contain preservatives such as, but not limited to,chlorobutanol and benzalkonium chloride. A surfactant may be present toenhance absorption of the subject proteins by the nasal mucosa. Asubject antibody can be administered as an injectable formulation.Typically, injectable compositions are prepared as liquid solutions orsuspensions; solid forms suitable for solution in, or suspension in,liquid vehicles prior to injection may also be prepared. The preparationmay also be emulsified or the antibody encapsulated in liposomevehicles.

Suitable excipient vehicles are, for example, water, saline, dextrose,glycerol, ethanol, or the like, and combinations thereof. In addition,if desired, the vehicle may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents or pH buffering agents.Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in the art. See, e.g., Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17thedition, 1985. The composition or formulation to be administered will,in any event, contain a quantity of a subject antibody adequate toachieve the desired state in the subject being treated.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

In some embodiments, a subject antibody is formulated in a controlledrelease formulation. Sustained-release preparations may be preparedusing methods well known in the art. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody in which the matrices arein the form of shaped articles, e.g. films or microcapsules. Examples ofsustained-release matrices include polyesters, copolymers of L-glutamicacid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,hydrogels, polylactides, degradable lactic acid-glycolic acid copolymersand poly-D-(−)-3-hydroxybutyric acid. Possible loss of biologicalactivity and possible changes in immunogenicity of antibodies comprisedin sustained-release preparations may be prevented by using appropriateadditives, by controlling moisture content and by developing specificpolymer matrix compositions. Controlled release within the scope of thisinvention can be taken to mean any one of a number of extended releasedosage forms. The following terms may be considered to be substantiallyequivalent to controlled release, for the purposes of the presentinvention: continuous release, controlled release, delayed release,depot, gradual release, long-term release, programmed release, prolongedrelease, proportionate release, protracted release, repository, retard,slow release, spaced release, sustained release, time coat, timedrelease, delayed action, extended action, layered-time action, longacting, prolonged action, repeated action, slowing acting, sustainedaction, sustained-action medications, and extended release. Furtherdiscussions of these terms may be found in Lesczek Krowczynski,Extended-Release Dosage Forms, 1987 (CRC Press, Inc.).

The various controlled release technologies cover a very broad spectrumof drug dosage forms. Controlled release technologies include, but arenot limited to physical systems and chemical systems.

Physical systems include, but are not limited to, reservoir systems withrate-controlling membranes, such as microencapsulation,macroencapsulation, and membrane systems; reservoir systems withoutrate-controlling membranes, such as hollow fibers, ultra microporouscellulose triacetate, and porous polymeric substrates and foams;monolithic systems, including those systems physically dissolved innon-porous, polymeric, or elastomeric matrices (e.g., nonerodible,erodible, environmental agent ingression, and degradable), and materialsphysically dispersed in non-porous, polymeric, or elastomeric matrices(e.g., nonerodible, erodible, environmental agent ingression, anddegradable); laminated structures, including reservoir layers chemicallysimilar or dissimilar to outer control layers; and other physicalmethods, such as osmotic pumps, or adsorption onto ion-exchange resins.

Chemical systems include, but are not limited to, chemical erosion ofpolymer matrices (e.g., heterogeneous, or homogeneous erosion), orbiological erosion of a polymer matrix (e.g., heterogeneous, orhomogeneous). Additional discussion of categories of systems forcontrolled release may be found in Agis F. Kydonieus, Controlled ReleaseTechnologies: Methods, Theory and Applications, 1980 (CRC Press, Inc.).

There are a number of controlled release drug formulations that aredeveloped for oral administration. These include, but are not limitedto, osmotic pressure-controlled gastrointestinal delivery systems;hydrodynamic pressure-controlled gastrointestinal delivery systems;membrane permeation-controlled gastrointestinal delivery systems, whichinclude microporous membrane permeation-controlled gastrointestinaldelivery devices; gastric fluid-resistant intestine targetedcontrolled-release gastrointestinal delivery devices; geldiffusion-controlled gastrointestinal delivery systems; andion-exchange-controlled gastrointestinal delivery systems, which includecationic and anionic drugs. Additional information regarding controlledrelease drug delivery systems may be found in Yie W. Chien, Novel DrugDelivery Systems, 1992 (Marcel Dekker, Inc.). Some of these formulationswill now be discussed in more detail.

Dosages

A suitable dosage can be determined by an attending physician or otherqualified medical personnel, based on various clinical factors. As iswell known in the medical arts, dosages for any one patient depend uponmany factors, including the patient's size, body surface area, age, theparticular compound to be administered, sex of the patient, time, androute of administration, general health, and other drugs beingadministered concurrently. A subject antibody may be administered inamounts between 1 ng/kg body weight and 20 mg/kg body weight per dose,e.g. between 0.1 mg/kg body weight to 10 mg/kg body weight, e.g. between0.5 mg/kg body weight to 5 mg/kg body weight; however, doses below orabove this exemplary range are envisioned, especially considering theaforementioned factors. If the regimen is a continuous infusion, it canalso be in the range of 1 μg to 10 mg per kilogram of body weight perminute.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific antibody, the severity of the symptoms and thesusceptibility of the subject to side effects.

Preferred dosages for a given compound are readily determinable by thoseof skill in the art by a variety of means.

Routes of Administration

A subject antibody is administered to an individual using any availablemethod and route suitable for drug delivery, including in vivo and exvivo methods, as well as systemic and localized routes ofadministration.

Conventional and pharmaceutically acceptable routes of administrationinclude intranasal, intramuscular, intratracheal, subcutaneous,intradermal, topical application, intravenous, intraarterial, rectal,nasal, oral, and other enteral and parenteral routes of administration.Routes of administration may be combined, if desired, or adjusteddepending upon the antibody and/or the desired effect. A subjectantibody composition can be administered in a single dose or in multipledoses. In some embodiments, a subject antibody composition isadministered orally. In some embodiments, a subject antibody compositionis administered via an inhalational route. In some embodiments, asubject antibody composition is administered intranasally. In someembodiments, a subject antibody composition is administered locally. Insome embodiments, a subject antibody composition is administeredintracranially. In some embodiments, a subject antibody composition isadministered intravenously.

The agent can be administered to a host using any available conventionalmethods and routes suitable for delivery of conventional drugs,including systemic or localized routes. In general, routes ofadministration contemplated by the invention include, but are notnecessarily limited to, enteral, parenteral, or inhalational routes.

Parenteral routes of administration other than inhalation administrationinclude, but are not necessarily limited to, topical, transdermal,subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal,intrasternal, and intravenous routes, i.e., any route of administrationother than through the alimentary canal. Parenteral administration canbe carried to effect systemic or local delivery of a subject antibody.Where systemic delivery is desired, administration typically involvesinvasive or systemically absorbed topical or mucosal administration ofpharmaceutical preparations.

A subject antibody can also be delivered to the subject by enteraladministration. Enteral routes of administration include, but are notnecessarily limited to, oral and rectal (e.g., using a suppository)delivery.

By treatment is meant at least an amelioration of the symptomsassociated with the pathological condition afflicting the host, whereamelioration is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, e.g. symptom, associated with thepathological condition being treated, such as a B cell malignancy or Bcell-mediated autoimmune disorder. As such, treatment also includessituations where the pathological condition, or at least symptomsassociated therewith, are completely inhibited, e.g. prevented fromhappening, or stopped, e.g. terminated, such that the host no longersuffers from the pathological condition, or at least the symptoms thatcharacterize the pathological condition.

In some embodiments, a subject antibody is administered by injection,e.g., for systemic delivery (e.g., intravenous infusion) or to a localsite.

A variety of hosts (wherein the term “host” is used interchangeablyherein with the terms “subject,” “individual,” and “patient”) aretreatable according to the subject methods. Generally such hosts are“mammals” or “mammalian,” where these terms are used broadly to describeorganisms which are within the class mammalia, including the orderscarnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, andrats), and primates (e.g., humans, chimpanzees, and monkeys). In someembodiments, the hosts will be humans.

Kits with unit doses of a subject antibody, e.g. in oral or injectabledoses, are provided. In such kits, in addition to the containerscontaining the unit doses will be an informational package insertdescribing the use and attendant benefits of the antibody in treatingpathological condition of interest. Preferred compounds and unit dosesare those described herein above.

Treatment Methods

The present disclosure provides methods of treating a disease ordisorder associated with or caused by a CD22-positive B cell, e.g., acancerous CD22-positive B cell; an autoreactive CD22-positive B cell.

Treating B Cell Malignancies

The present disclosure provides methods of treating a B cell malignancy,the methods generally involving administering to an individual in needthereof (e.g., an individual having a B cell malignancy) an effectiveamount of a subject antibody, alone (e.g., in monotherapy) or incombination (e.g., in combination therapy) with one or more additionaltherapeutic agents. B-cell malignancies include, e.g., non-Hodgkin'slymphoma, Burkitt's lymphoma, multiple myeloma, chronic lymphocyticleukemia, hairy cell leukemia and prolymphocytic leukemia. In someembodiments, an effective amount of a subject antibody is an amountthat, when administered alone (e.g., in monotherapy) or in combination(e.g., in combination therapy) with one or more additional therapeuticagents, in one or more doses, is effective to reduce the number ofcancerous B cells in an individual by at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90%, ormore, compared to the number of cancerous B cells in the individual inthe absence of treatment with the antibody.

Combination Therapy

In some embodiments, a subject method of treating a B cell malignancyinvolves administering a subject antibody and one or more additionaltherapeutic agents. Suitable additional therapeutic agents include, butare not limited to, a cancer chemotherapeutic agent (as describedabove).

Treating B Cell-Mediated Autoimmune Disorders

The present disclosure provides methods of treating a B cell-mediatedautoimmune disorder, the methods generally involving administering to anindividual in need thereof (e.g., an individual having a B cell-mediatedautoimmune disorder) an effective amount of a subject antibody, alone(e.g., in monotherapy) or in combination (e.g., in combination therapy)with one or more additional therapeutic agents. B cell-mediatedautoimmune disorders are autoimmune disorders in which the pathology isprimarily due to the presence of antibody specific for one or moreautoantigens. As such, a B cell-mediated autoimmune disorder can also bereferred to as an antibody-mediated autoimmune disorder.

B cell-mediated autoimmune disorders include, e.g., systemic lupuserythematosus, myasthenia gravis, autoimmune myocarditis, rheumatoidarthritis, and the like.

In some embodiments, an effective amount of a subject antibody is anamount that, when administered alone (e.g., in monotherapy) or incombination (e.g., in combination therapy) with one or more additionaltherapeutic agents, in one or more doses, is effective to reduce thenumber of autoreactive B cells (B cells producing autoantibody) in anindividual by at least about 5%, at least about 10%, at least about 15%,at least about 20%, at least about 25%, at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, or more, compared to the numberof autoreactive B cells in the individual in the absence of treatmentwith the antibody.

Combination Therapy

In some embodiments, a subject method of treating a B cell-mediatedautoimmune disease involves administering a subject antibody and one ormore additional therapeutic agents. Suitable additional therapeuticagents include, but are not limited to, immunosuppressive agents,anti-inflammatory agents, and the like.

Subjects Suitable for Treatment

A variety of subjects are suitable for treatment with a subject method.Suitable subjects include any individual, e.g., a human, who has a Bcell malignancy; who has been diagnosed with a B cell malignancy; whohas had a B cell malignancy and is at risk for recurrence of the B cellmalignancy; who has been treated for a B cell malignancy with an agentother than a subject anti-CD22 antibody (e.g., who has been treated witha cancer chemotherapeutic agent) and who has not responded to the agent;or who has been treated for a B cell malignancy with an agent other thana subject anti-CD22 antibody (e.g., who has been treated with a cancerchemotherapeutic agent) and who initially responded to the agent butsubsequently ceased to respond (e.g., relapsed).

Subjects who are suitable for treatment for a B cell-mediated autoimmunedisorder using a subject method include any individual e.g., a human,who has a B cell-mediated autoimmune disorder; who has been diagnosedwith a B cell-mediated autoimmune disorder; who has had a Bcell-mediated autoimmune disorder and is at risk for recurrence of the Bcell-mediated autoimmune disorder; who has been treated for a Bcell-mediated autoimmune disorder with an agent other than a subjectanti-CD22 antibody (e.g., who has been treated with an immunosuppressiveagent) and who has not responded to the agent; or who has been treatedfor a B cell-mediated autoimmune disorder with an agent other than asubject anti-CD22 antibody (e.g., who has been treated with animmunosuppressive agent) and who initially responded to the agent butsubsequently ceased to respond (e.g., relapsed).

Detection Methods

The present disclosure provides various detection methods that involveuse of a subject antibody. Detection methods include diagnostic methods,prognostic methods, and monitoring methods. A subject detection methodgenerally involves detecting CD22 positive cells, e.g., B cells, e.g.,cancerous B cells.

In some embodiments, a subject method is a diagnostic method, e.g., todetermine whether an individual has a B cell malignancy.

In some embodiments, a subject method is a monitoring method, e.g., anindividual who has been diagnosed as having a B cell malignancy, and isbeing treated for the disorder, is monitored for response to thetreatment and/or progression/regression of the disorder.

In some cases, a subject detection method involves administering to anindividual a detectably labeled anti-CD22 antibody of the presentdisclosure; and detecting binding of the antibody to tissues in theindividual. Detection can be achieved, e.g., by magnetic resonanceimaging or other suitable imaging technique.

In other instances, a subject detection method involves contacting adetectably labeled anti-CD22 antibody of the present disclosure with abiological sample obtained from an individual; and detecting binding ofthe antibody to molecules in the biological sample.

The anti-CD22 antibody can be labeled directly or indirectly. Indirectlabels include a secondary antibody that comprises a detectable label,where the secondary antibody binds a subject anti-CD22 antibody. Otherindirect labels include biotin, where a biotinylated anti-CD22 antibodycan be detected using avidin or streptavidin that comprises a detectablelabel.

Suitable detectable labels include any composition detectable byspectroscopic, photochemical, biochemical, immunochemical, electrical,optical or chemical means. Suitable include, but are not limited to,magnetic beads (e.g. Dynabeads™), fluorescent dyes (e.g., fluoresceinisothiocyanate, texas red, rhodamine, a green fluorescent protein, a redfluorescent protein, a yellow fluorescent protein, and the like),radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g.,horseradish peroxidase, alkaline phosphatase, luciferase, and otherscommonly used in an enzyme-linked immunosorbent assay (ELISA)), andcolorimetric labels such as colloidal gold or colored glass or plastic(e.g. polystyrene, polypropylene, latex, etc.) beads.

In some embodiments, a subject antibody comprises a contrast agent or aradioisotope, where the contrast agent or radioisotope is one that issuitable for use in imaging, e.g., imaging procedures carried out onhumans. Non-limiting examples of labels include radioisotope such as¹²³¹I (iodine), ¹⁸F (fluorine), ⁹⁹Tc (technetium), ¹¹¹In (indium), and⁶⁷Ga (gallium), and contrast agent such as gadolinium (Gd), dysprosium,and iron. Radioactive Gd isotopes (¹⁵³Gd) also are available andsuitable for imaging procedures in non-human mammals. A subject antibodycan be labeled using standard techniques. For example, a subjectantibody can be iodinated using chloramine T or1,3,4,6-tetrachloro-3α,6α-dephenylglycouril. For fluorination, fluorineis added to a subject antibody during the synthesis by a fluoride iondisplacement reaction. See, Muller-Gartner, H., TIB Tech., 16:122-130(1998) and Saji, H., Crit. Rev. Ther. Drug Carrier Syst., 16(2):209-244(1999) for a review of synthesis of proteins with such radioisotopes. Asubject antibody can also be labeled with a contrast agent throughstandard techniques. For example, a subject antibody can be labeled withGd by conjugating low molecular Gd chelates such as Gd diethylenetriamine pentaacetic acid (GdDTPA) or Gdtetraazacyclododecanetetraacetic (GdDOTA) to the antibody. See, Caravanet al., Chem. Rev. 99:2293-2352 (1999) and Lauffer et al., J. Magn.Reson. Imaging, 3:11-16 (1985). A subject antibody can be labeled withGd by, for example, conjugating polylysine-Gd chelates to the antibody.See, for example, Curtet et al., Invest. Radiol., 33(10):752-761 (1998).Alternatively, a subject antibody can be labeled with Gd by incubatingparamagnetic polymerized liposomes that include Gd chelator lipid withavidin and biotinylated antibody. See, for example, Sipkins et al.,Nature Med., 4:623-626 (1998).

Suitable fluorescent proteins that can be linked to a subject antibodyinclude, but are not limited to, a green fluorescent protein fromAequoria victoria or a mutant or derivative thereof e.g., as describedin U.S. Pat. Nos. 6,066,476; 6,020,192; 5,985,577; 5,976,796; 5,968,750;5,968,738; 5,958,713; 5,919,445; 5,874,304; e.g., Enhanced GFP, manysuch GFP which are available commercially, e.g., from Clontech, Inc.; ared fluorescent protein; a yellow fluorescent protein; any of a varietyof fluorescent and colored proteins from Anthozoan species, as describedin, e.g., Matz et al. (1999) Nature Biotechnol. 17:969-973; and thelike.

Kits

The present disclosure provides a kit (e.g., a test kit) that includes asubject antibody. A subject kit is useful in carrying out a subjectdetection method.

A subject kit can include one or more of: a subject antibody, a nucleicacid encoding the same, or a cell comprising a subject nucleic acid. Thesubject antibody in a subject kit can be humanized. A subject kit caninclude reagents for labeling the antibody. In some embodiments, theantibody in a subject kit comprises a detectable label.

Other optional components of the kit include: a buffer; a proteaseinhibitor; a detectable label; etc. Where a subject kit comprises asubject nucleic acid, the nucleic acid may also have restrictions sites,multiple cloning sites, primer sites, etc. The various components of thekit may be present in separate containers or certain compatiblecomponents may be pre-combined into a single container, as desired.

In addition to above-mentioned components, a subject kit can includeinstructions for using the components of the kit to practice a subjectmethod. The instructions for practicing a subject method are generallyrecorded on a suitable recording medium. For example, the instructionsmay be printed on a substrate, such as paper or plastic, etc. As such,the instructions may be present in the kits as a package insert, in thelabeling of the container of the kit or components thereof (i.e.,associated with the packaging or subpackaging) etc. In otherembodiments, the instructions are present as an electronic storage datafile present on a suitable computer readable storage medium, e.g.compact disc-read only memory (CD-ROM), digital versatile disk (DVD),diskette, etc. In yet other embodiments, the actual instructions are notpresent in the kit, but means for obtaining the instructions from aremote source, e.g. via the internet, are provided. An example of thisembodiment is a kit that includes a web address where the instructionscan be viewed and/or from which the instructions can be downloaded. Aswith the instructions, this means for obtaining the instructions isrecorded on a suitable substrate.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Commercially available reagents referred to in the Examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of cells identified in the Examples and throughout thespecification by ECACC accession numbers is the European Collection ofCell Cultures (ECACC), Salisbury, England. Unless otherwise defined, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. Exemplary methods and materials are described belowalthough methods and materials similar or equivalent to those describedherein can also be used in the practice or testing of the presentinvention. The materials, methods, and examples are illustrative onlyand not intended to be limiting in scope.

Example 1—Generation of Chimeric Antibody

The heavy and light chain variable (V) region sequences of the mouseRFB4 monoclonal antibody (Campana. et al (1985) J. Immunol., 134,1524-1530. Mansfield et at (1997) Blood, 90, 2020-2026) were synthesizedand subcloned into pANT antibody expression vectors (FIG. 1) with heavyand light chain V regions cloned into pANT17 and pANT13 respectively.Heavy chain V region genes were cloned into pANT17 via MluI and HindIIIsites in frame with the human γ1 heavy chain gene (G1m3 (G1m(f))allotype) and light chain V region genes were cloned into pANT13 viaBssHII and BamHI sites in frame with the human kappa light chainconstant region gene (Km3 allotype). Transcription of both heavy andlight chain genes was under the control of the CMV I/E promoter (U.S.Pat. No. 5,168,062 and U.S. Pat. No. 5,385,839, University of Iowa) andthe pANT17 plasmid contained a mutant dhfr minigene (Simonsen & Levinson1983, Proc. Natl. Acad. Sci. USA 80:2495-2499) under the control of aSV40 promoter and polyA sequence for selection in eukaryotic cells. BothpANT17 and pANT13 contained a β-lactamase (Ap^(R)) gene for prokaryoticselection and a pMB1 origin of replication for propagation inprokaryotic cells. All plasmids were propagated in E. coli DH5 alpha(Invitrogen Cat. No. 18265-017). The heavy and light chain expressionconstructs were subsequently co-transfected either transiently intoHEK293 c18 cells by calcium phosphate-based transfection or stablytransfected into NS0 cells by electroporation. The resulting chimericRFB4 antibody as secreted from the HEK293 c18 or NS0 cells was purifiedfrom the cell culture supernatants by Protein A chromatography anddesalted into phosphate-buffered saline (PBS) using PD-10 columns (GEHealthcare Cat. No. 17-0851-01). Concentrations were determined by UVabsorbance at 280 nm using a molar extinction coefficient based on theamino acid composition of each individual antibody.

Example 2—Generation of Humanized Antibodies

Humanized antibodies were generated using methods described in EP1844074(Antitope Ltd). Structural models of the mouse RFB4 V regions wereproduced using Swiss PDB and analyzed in order to identify importantframework amino acids that were likely to be important for the CD22binding properties of the antibody (‘constraining residues’). A databaseof human V region sequences was used to identify segments of human Vregion sequences containing each of the constraining residues to be usedin design of the humanized antibodies. RFB4 CDR sequences were retainedin the designed humanized antibody sequences. A set of favored V regionsequences were designed and analyzed for the prediction of non-germlinemajor histocompatibility complex (MHC) class II peptide binding by insilico analysis as described in Fothergill et al. (WO9859244, assigneeEclagen Ltd) and also for known CD4⁺ T-cell epitopes using databasesincluding “The Immune Epitope Database and Analysis Resource”,(http://www(dot)immunepitope(dot)org/). V region sequences withpredicted non-germline MHC class II binding peptides or with significanthits against T cell epitope databases were discarded. This resulted in areduced set of V region sequences. Selected heavy and light chain Vregion sequences were then combined to produce humanized heavy and lightchain variable region amino acid sequences. Six heavy chains and fourlight chain sequences (designated VH1 to VH6, and Vκ1 to Vκ4respectively) were selected for use in producing humanized RFB4antibodies. Heavy chains comprising VH sequences VH3 (SEQ ID NO:3), VH4(SEQ ID NO:4), and VH5 (SEQ ID NO:5), VH6 (SEQ ID NO:6) were paired withlight chains comprising VL sequences VK1 (SEQ ID NO:7), VK2 (SEQ IDNO:8), and VK4 (SEQ ID NO:9). DNA encoding humanized VH and VK variantswere synthesized and subcloned into the expression vectors pANT17 andpANT13 (FIG. 1) as described in Example 1. All combinations of humanizedVH and Vκ chains were transiently transfected into HEK293 c18 cells andantibody was purified by protein A chromatography from the culturesupernatants followed by desalting as described in Example 1.

Example 3—Analysis of Humanised Antibodies

The binding of HEK-derived RFB4 humanized variants to CD22 antigen wasassessed in a competition enzyme-linked immunosorbent assay (ELISA)against the parent chimeric antibody. The parental RFB4 chimericantibody was biotinylated using Biotin Tag™ Micro Biotinylation kit(Sigma-Aldrich). 96 well MaxiSorp plates (Nunc) were coated with 1.0μg/ml CD22-Fc (R&D Systems Cat. No. 1968SL) in Dulbecco's PBS (PAALaboratories, Yeovil, UK) (60 μl final volume) at 4° C. overnight.Plates were blocked with Dulbecco's PBS-2% BSA for 1 hour at roomtemperature. Plates were washed 3 times with wash buffer (0.05% Tween20in Dulbecco's-PBS). Test humanized antibodies at various concentrationswere premixed with biotinylated parent chimeric antibody (0.04 μg/mlfinal concentration) and then added to the CD22-Fc plate (60 μl finalvolume). All samples were tested in duplicate. Plates were incubated for1 h at room temperature and washed 3 times with wash buffer. 60 μl of a1 in 1000 dilution of Streptavidin horse radish peroxidase (HRP)(Sigma-Aldrich) was added and incubated for 1 hour at room temperature.Plates were washed 3 times with wash buffer and 60 μl of3,3′,5,5′-tetramethybenzidine (TMB) substrate (Invitrogen) was added andincubated at room temperature in the dark to allow the colour todevelop. The reaction was stopped by adding 50 μl of 3M HCl. Plates wereread at 450 nm using Dynex plate reader.

As shown in FIG. 2, all lead humanized RFB4 variants displayedcompetitive binding profiles similar to the parent chimeric antibody.The IC₅₀ of chimeric RFB4 and humanized RFB4 antibodies, relative towild-type RFB4 IgG1 antibody, is shown in Table 2, below.

TABLE 2 IC₅₀ relative to Construct Chimeric RFB4 IgG1 Chimeric RFB4 1.0VH3/VK1 0.8 VH3/VK2 1.0 VH3/VK4 1.1 VH4/VK1 0.9 VH4/VK2 1.1 VH4/VK4 1.0VH5/VK1 0.9 VH5/VK2 1.0 VH5/VK4 1.0 VH6/VK1 0.9 VH6/VK2 1.0 VH6/VK4 1.0

Example 4—Analysis of CD4+ T Cell Responses

Peripheral blood mononuclear cells (PBMC) were isolated from healthycommunity donor buffy coats (from blood drawn within 24 hours) obtainedfrom the UK National Blood Transfusion Service (Addenbrooke's Hospital,Cambridge, UK) and according to approval granted by Addenbrooke'sHospital Local Research Ethics Committee. PBMC were isolated from buffycoats by Lymphoprep (Axis-shield, Dundee, UK) density centrifugation andCD8⁺ T cells were depleted using CD8⁺ RosetteSep™ (StemCell TechnologiesInc, London, UK). Donors were characterized by identifying HLA-DRhaplotypes using an HLA single-specific-primer polymerase chain reaction(SSP-PCR) based tissue-typing kit (Biotest, Solihull, UK). T cellresponses to a ‘reproducibility’ control antigen (Keyhole LimpetHaemocyanin (KLH), Pierce (Perbio), Cramlington, UK) as well as controlpeptides derived from Influenza A and Epstein Barr viruses were alsodetermined. PBMC were then frozen and stored in liquid nitrogen untilrequired.

The chimeric and lead VH4/VK1, VH5/VK1, VH6/VK4 humanized antibodieswere purified from transiently transfected HEK293 c18 cell lines byProtein A chromatography followed by size exclusion chromatography usinga 26/60 Superdex 5200 column (GE Healthcare) in 1×PBS. Monomeric peakfractions were collected, quantified and endotoxin levels analyzed forall preparations using the Endosafe®-PTS™ (Charles River, Margate, UK)system.

A cohort of 20 donors was selected to best represent the number andfrequency of HLA-DR allotypes expressed in the world population.Analysis of the allotypes expressed in the cohort against thoseexpressed in the world population revealed that all major HLA-DR alleles(individual allotypes with a frequency>5% expressed in the worldpopulation) were well represented. PBMCs from each donor were revived inAIM-V® culture medium (Invitrogen, Paisley, UK), washed and resuspendedin AIM-V® to 4-6×10⁶ PBMC/ml. For each donor, bulk cultures wereestablished in which 1 ml proliferation cell stock was added to theappropriate wells of a 24 well plate. 0.5 ml culture medium togetherwith 0.5 ml of each diluted test sample were added to the PBMC to give afinal concentration of 50 μg/ml per sample. For each donor, areproducibility control (cells incubated with 100 μg/ml KLH) and aculture medium only well were also included. Cultures were incubated fora total of 8 days at 37° C. with 5% CO₂. On days 5, 6, 7 and 8, thecells in each well were gently resuspended and 3×100 μl aliquotstransferred to each well of a round bottomed 96 well plate. The cultureswere pulsed with 0.75 μCi [³H]-Thymidine (Perkin Elmer®, Beaconsfield,UK) in 100 μl AIM-V® culture medium and incubated for a further 18 hoursbefore harvesting onto filter mats (Perkin Elmer®) using a TomTec MachIII cell harvester. Counts per minute (cpm) for each well weredetermined by Meltilex™ (Perkin Elmer®) scintillation counting on a 1450Microbeta Wallac Trilux Liquid Scintillation Counter (Perkin Elmer®) inparalux, low background counting.

For proliferation assays, an empirical threshold of a Stimulation Index(SI) equal to or greater than 2 (SI≥2.0) has been previously establishedwhereby samples inducing proliferative responses above this thresholdare deemed positive (where included, borderline SIs≥1.90 arehighlighted). For proliferation data sets (n=3), positive responses weredefined by statistical and empirical thresholds:

-   -   1) Significance (p<0.05) of the response by comparing cpm of        test wells against medium control wells using unpaired two        sample student's t-test.    -   2) SI equal to or greater than 2 (SI≥2.0).    -   3) Basal cpm>150 cpm

In addition, intra-assay variation was assessed by calculating the CVsand SDs of the raw data from replicate cultures.

FIGS. 3A-D depict healthy donor T cell proliferation responses to testantibodies. PBMC from bulk cultures were sampled and assessed forproliferation on days 5, 6, 7, and 8 after incubation with the threetest samples. Proliferation responses with an SI≥2.0 (p<0.05), indicatedby the dotted line that were significant (p<0.05) using an unpaired, twosample Student's t test were considered positive.

The data are shown in FIGS. 3A-D, and are summarized in Table 3, below.FIGS. 3A-D illustrate the donor SI responses to each of the testantibodies throughout the time course. The fully humanised anti-CD22antibodies (VH4/VK1, VH5/VK1, VH6/VK4) induced no positive responsesusing SI≥2.0, p<0.05 threshold in any of the donors in the proliferationassay, whereas the chimeric anti-CD22 antibody induced positive T cellproliferation responses in 25% of donors. FIG. 3A: chimeric antibody;FIG. 3B: VH4/VK1; FIG. 3C: VH5/VK1; FIG. 3D: VH6/VK4.

TABLE 3 Anti-CD22 Chimeric VH4/VK1 VH5/VK1 VH6/VK4 KLH Donor 1 P Donor 2Donor 3  P* Donor 4 Donor 5 Donor 6 Donor 7 P P Donor 8 P Donor 9 PDonor 10 Donor 11 P Donor 12 P Donor 13 P Donor 14 P Donor 15 P Donor 16P P Donor 17 P Donor 18 P P Donor 19 P P Donor 20 P Proliferation % 25 00 0 70

Table 3. Summary of healthy donor proliferation. Positive proliferation(“P”) responses from days 5-8 (SI≥2.0, significant p<0.05) throughoutthe time course (SI≥2.0, significant p<0.05). Borderline responses(significant p<0.05 with SI≥1.90) for proliferation (P*) are shown. Thetotal frequency of response in the donor cohort is shown as apercentage.

Example 5—Analysis of Internalization

Raji cells, grown to ˜0.5×10⁶ cells/mL, were used at 0.35-0.5×10⁶ cellsper test. Cells were resuspended at 100 μL/tube in phosphate bufferedsaline+1% fetal calf serum (FCS; Buffer A). Controls included cellsexposed only to 4° C. or to both 4° and 37° C. that were incubated withno antibody or with secondary antibody only. Primary antibody was addedat 1 μg/tube and cells were incubated on ice for 30 min. Then, cellswere washed: 1 mL Buffer A was added, cells were gently pelleted bycentrifugation, and supernatant was removed. This was repeated for atotal of 2 washes. Cells to be exposed to 37° C. were resuspended in 37°C. RMPI+10% fetal calf serum (FCS), 2 mM glutamine, and incubated at 37°C., 5% CO₂ for 45 min. Next, cells were washed one time in 1 mL ice-coldBuffer A. Secondary antibody (fluorescein-conjugated goat anti-human Fc;Jackson Immunoresearch, West Grove, Pa.) was added at a 1:100 dilutionin Buffer A and cells were incubated on ice for 30 min. Finally, cellswere washed twice in ice-cold Buffer A and resuspended in 300 μL BufferA. Cells were kept in the dark at 4° C. and analyzed within 18 h by flowcytometry on a Becton Dickinson FACSCanto using FACSDiva software. Datawere analyzed as follows: the mean fluorescence intensity (MFI) of thesecondary antibody on the FL1 channel (used to detect fluorescein) wassubtracted from the MFI of the signals generated when primary antibodieswere included. The resulting values were then termed the MFI signals forthe current experiment. The MFI values of cells held at 4° C.represented the binding of the antibody to the cell surface. The MFIvalues of cells exposed to 37° C. represented the signal generated byantibody remaining at the cell surface after internalization of CD22-Abcomplexes. The difference between the MFI at 4° and 37° C. correspondsto the internalization of bound antibody (FIG. 4). To allow forcomparison among experiments performed on different days, binding andinternalization values were sometimes normalized to the wild-typeantibody, RFB4.

Example 6—Analysis of CD22 Binding Affinity

Human CD22 was obtained from either from R&D Systems (huCD22-Fc form,catalog #1968-SL-050) or from Sino Biological Inc (huCD22 with His tag,catalog #11958-H08H) and was biotinylated to Lys using a LC-biotin. Thebinding of variants 12 through 20 to huCD22 was measured using aForteBio instrument. StreptAvidin derivatized ForteBio biosensors wereloaded with the biotinylated huCD22 molecules. The concentrations of theantibodies of interest were confirmed by A₂₈₀ and loaded biosensors wereexposed to increasing concentrations of each of the antibodies. Thekinetic association and dissociation rates were determined toward thehuCD22 at 4-5 concentrations at pH 7.25 and the K_(D)'s were determined(FIG. 5).

Example 7—Analysis of Aggregation

To determine the extent of aggregation of the RFB4 variants, 20 μg ofantibody was analyzed using size-exclusion high-performance liquidchromatography (Tosoh #08541 G300 SW_(XL) 7.8 mm×30 cm; mobile phase 25mM sodium phosphate buffer, 300 mM NaCl, pH 6.8; 0.8 mL/min; monitorabsorbance at 220 nm and 280 nm). Results are highlighted in FIGS.6A-6D.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A composition comprising (i) an antibody thatspecifically binds an epitope in CD22, wherein the antibody comprises:a) an immunoglobulin heavy chain comprising a VH region having the aminoacid sequenceEVQLVESGGGLVKPGGSLX₁LSCAASGFAFSIYDMSWVRQAPGKGLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNX₂LYLQMX₃SLRAEDTAMYYCARHSGYGSSYGVLFAYWG QGTLVTVSS(SEQ ID NO:1), where X₁ is K or R; X₂ is S or T; and X₃ is N or S; andb) an immunoglobulin light chain; and (ii) a carrier.
 2. The compositionof claim 1, wherein the immunoglobulin light chain comprises the aminoacid sequence (SEQ ID NO: 7; VK1)DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTSILHSGVPSRFSGSGSGTDYTLTISSLQQEDFATYFCQQGNTL PWTFGGGTKVEIK.


3. The composition of claim 1, wherein the immunoglobulin light chaincomprises the amino acid sequence (SEQ ID NO: 8; VK2)DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTSILHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTL PWTFGGGTKVEIK.


4. The composition of claim 1, wherein the immunoglobulin light chaincomprises the amino acid sequence (SEQ ID NO: 9; VK4)DIQMTQSPSSVSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTL PWTFGGGTKVEIK.


5. A composition comprising (i) an antibody that specifically binds anepitope in CD22, wherein the antibody comprises: a) an immunoglobulinlight chain comprising the amino acid sequenceDIQMTQSPSSX₁SASVGDRVTITCRASQDISNYLNWYQQKPGKAX₂KLLIYYTSILHSGVPSRFSGSGSGTDYTLTISSLQX₃EDFATYFCQQGNTLPWTFGGGTKVEIK (SEQ ID NO:2), whereX₁ is L (Leu) or V (Val); X₂ is V or P; and X₃ is Q or P; and b) animmunoglobulin heavy chain; and (ii) a carrier.
 6. The composition ofclaim 5, wherein the immunoglobulin heavy chain comprises an amino acidsequence selected from: (SEQ ID NO: 3; VH3)EVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQAPGKGLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNTLYLQMSSLRAEDTAMYYCARHSGYGSSYGVLFAYWGQGTLVTVSS; (SEQ ID NO: 4; VH4)EVQLVESGGGLVKPGGSLRLSCAASGFAFSIYDMSWVRQAPGKGLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNSLYLQMSSLRAEDTAMYYCARHSGYGSSYGVLFAYWGQGTLVTVSS; (SEQ ID NO: 5; VH5)EVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQAPGKGLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAMYYCARHSGYGSSYGVLFAYWGQGTLVTVSS; and (SEQ ID NO: 6; VH6)EVQLVESGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQAPGKGLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNSLYLQMSSLRAEDTAMYYCARHSGYGSSYGVLFAYWGQGTLVTVSS.


7. The composition of claim 5, wherein the light chain region and theheavy chain region are present in separate polypeptides.
 8. Thecomposition of claim 5, wherein the light chain region and the heavychain region are present in a single polypeptide.
 9. The composition ofclaim 5, wherein the antibody binds the epitope with an affinity of fromabout 10⁷M⁻¹ to about 10¹²M⁻¹.
 10. The composition of claim 5, whereinthe heavy chain region is of the isotype IgG1, IgG2, IgG3, or IgG4. 11.The composition of claim 5, wherein the antibody is detectably labeled.12. The composition of claim 5, wherein the antibody is a F_(v),scF_(v), F_(ab), F(_(ab′))₂, or F_(ab′).
 13. The composition of claim 5,wherein the antibody comprises a covalently linked non-peptide syntheticpolymer.
 14. The composition of claim 13, wherein the synthetic polymeris poly(ethylene glycol) polymer.
 15. The composition of claim 5,wherein the antibody comprises a covalently linked lipid or fatty acidmoiety.
 16. The composition of claim 5, wherein the antibody comprises acovalently linked polysaccharide or carbohydrate moiety.
 17. Thecomposition of claim 5, wherein the antibody comprises a contrast agent.18. The composition of claim 5, wherein the antibody comprises anaffinity domain.
 19. The composition of claim 5, wherein the antibody isimmobilized on a solid support.
 20. The composition of claim 5, whereinthe antibody is a single chain F_(v) (scF_(v)) antibody.
 21. Thecomposition of claim 20, wherein the scF_(v) is multimerized.
 22. Thecomposition of claim 5, wherein the antibody comprises a covalentlylinked cytotoxin.
 23. The composition of claim 5, wherein the antibodycomprises a constant region amino acid sequence comprising an amino acidsequence of a sulfatase motif.
 24. The composition of claim 5, whereinthe antibody comprises a constant region amino acid sequence comprisingan amino acid sequence of a sulfatase motif, and wherein the sulfatasemotif is modified to comprise a 2-formylglycine (FGly) moiety.
 25. Thecomposition of claim 24, wherein the antibody comprises a heterologousmoiety covalently linked to the antibody via the FGly moiety.
 26. Thecomposition of claim 25, wherein the heterologous moiety is selectedfrom a drug, a toxin, a detectable label, a water-soluble polymer, and asynthetic peptide.
 27. A pharmaceutical composition comprising: a) aneffective amount of the composition of claim 1; and b) apharmaceutically acceptable carrier.
 28. The pharmaceutical compositionof claim 27, wherein the antibody is encapsulated in a liposome.
 29. Apharmaceutical composition comprising: a) the composition of claim 5;and b) a pharmaceutically acceptable carrier.
 30. A method of treating Bcell malignancy in a subject, the method comprising: administering to asubject having cancer an effective amount of the pharmaceuticalcomposition of claim 29.